CRISPR-Cas component systems, methods and compositions for sequence manipulation

ABSTRACT

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to US provisional patent applications61/736,527, 61/748,427, 61/768,959, 61/791,409 and 61/835,931, allentitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONfiled on Dec. 12, 2012, Jan. 2, 2013, Feb. 25, 2013, Mar. 15, 2013 andJun. 17, 2013, respectively.

Reference is made to US provisional patent applications 61/758,468;61/769,046; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and61/828,130, each entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30,2013; Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6,2013 and May 28, 2013 respectively. Reference is also made to U.S.provisional patent application 61/835,936, 61/836,127, 61/836,101,61/836,080, 61/836,123 and 61/835,973 each filed Jun. 17, 2013.Reference is also made to U.S. provisional patent application 61/842,322and U.S. patent application Ser. No. 14/054,414, entitled CRISPR-CASSYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS filed onJul. 2, 2013 and Oct. 15, 2013 respectively.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under the NIH PioneerAward DP1MH100706, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as genome perturbation or gene-editing, that may usevector systems related to Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) and components thereof.

PARTIES TO A JOINT RESEARCH AGREEMENT

The claimed invention was made by, on behalf of, and/or in connectionwith one or more of the following parties to a joint research agreement:The Broad Institute, Inc., Massachusetts Institute of Technology, andPresident and Fellows of Harvard College. The agreement was in effect onand before the date the claimed invention was made, and the claimedinvention was made as a result of activities undertaken within the scopeof the agreement.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 31, 2015, isnamed 44790.15.2003.txt and is 308,602 bytes in size.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that are affordable, easyto set up, scalable, and amenable to targeting multiple positions withinthe eukaryotic genome.

SUMMARY OF THE INVENTION

There exists a pressing need for alternative and robust systems andtechniques for sequence targeting with a wide array of applications.This invention addresses this need and provides related advantages. TheCRISPR/Cas or the CRISPR-Cas system (both terms are used interchangeablythroughout this application) does not require the generation ofcustomized proteins to target specific sequences but rather a single Casenzyme can be programmed by a short RNA molecule to recognize a specificDNA target, in other words the Cas enzyme can be recruited to a specificDNA target using said short RNA molecule. Adding the CRISPR-Cas systemto the repertoire of genome sequencing techniques and analysis methodsmay significantly simplify the methodology and accelerate the ability tocatalog and map genetic factors associated with a diverse range ofbiological functions and diseases. To utilize the CRISPR-Cas systemeffectively for genome editing without deleterious effects, it iscritical to understand aspects of engineering and optimization of thesegenome engineering tools, which are aspects of the claimed invention.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a tracr mate sequence and one ormore insertion sites for inserting one or more guide sequences upstreamof the tracr mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aCRISPR enzyme complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the tracr mate sequence that ishybridized to the tracr sequence; and (b) a second regulatory elementoperably linked to an enzyme-coding sequence encoding said CRISPR enzymecomprising a nuclear localization sequence; wherein components (a) and(b) are located on the same or different vectors of the system. In someembodiments, component (a) further comprises the tracr sequencedownstream of the tracr mate sequence under the control of the firstregulatory element. In some embodiments, component (a) further comprisestwo or more guide sequences operably linked to the first regulatoryelement, wherein when expressed, each of the two or more guide sequencesdirect sequence specific binding of a CRISPR complex to a differenttarget sequence in a eukaryotic cell. In some embodiments, the systemcomprises the tracr sequence under the control of a third regulatoryelement, such as a polymerase III promoter. In some embodiments, thetracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofsequence complementarity along the length of the tracr mate sequencewhen optimally aligned. Determining optimal alignment is within thepurview of one of skill in the art. For example, there are publicallyand commercially available alignment algorithms and programs such as,but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie,Geneious, Biopython and SeqMan. In some embodiments, the CRISPR complexcomprises one or more nuclear localization sequences of sufficientstrength to drive accumulation of said CRISPR complex in a detectableamount in the nucleus of a eukaryotic cell. Without wishing to be boundby theory, it is believed that a nuclear localization sequence is notnecessary for CRISPR complex activity in eukaryotes, but that includingsuch sequences enhances activity of the system, especially as totargeting nucleic acid molecules in the nucleus. In some embodiments,the CRISPR enzyme is a type II CRISPR system enzyme. In someembodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments,the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophilus Cas9,and may include mutated Cas9 derived from these organisms. The enzymemay be a Cas9 homolog or ortholog. In some embodiments, the CRISPRenzyme is codon-optimized for expression in a eukaryotic cell. In someembodiments, the CRISPR enzyme directs cleavage of one or two strands atthe location of the target sequence. In some embodiments, the CRISPRenzyme lacks DNA strand cleavage activity. In some embodiments, thefirst regulatory element is a polymerase III promoter. In someembodiments, the second regulatory element is a polymerase II promoter.In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19,20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20nucleotides in length. In general, and throughout this specification,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors.” Commonexpression vectors of utility in recombinant DNA techniques are often inthe form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g. transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g. 1, 2,3, 4, 5, or more pol Ill promoters), one or more pol II promoters (e.g.1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters(e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.Examples of pol III promoters include, but are not limited to, U6 and H1promoters. Examples of pol II promoters include, but are not limited to,the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally withthe RSV enhancer), the cytomegalovirus (CMV) promoter (optionally withthe CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter. Also encompassed by the term “regulatory element” are enhancerelements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR ofHTLV-1 (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer;and the intron sequence between exons 2 and 3 of rabbit 3-globin (Proc.Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression desired, etc. A vectorcan be introduced into host cells to thereby produce transcripts,proteins, or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., clustered regularlyinterspersed short palindromic repeats (CRISPR) transcripts, proteins,enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

In one aspect, the invention provides a vector comprising a regulatoryelement operably linked to an enzyme-coding sequence encoding a CRISPRenzyme comprising one or more nuclear localization sequences. In someembodiments, said regulatory element drives transcription of the CRISPRenzyme in a eukaryotic cell such that said CRISPR enzyme accumulates ina detectable amount in the nucleus of the eukaryotic cell. In someembodiments, the regulatory element is a polymerase II promoter. In someembodiments, the CRISPR enzyme is a type II CRISPR system enzyme. Insome embodiments, the CRISPR enzyme is a Cas9 enzyme. In someembodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.thermophilus Cas9, and may include mutated Cas9 derived from theseorganisms. In some embodiments, the CRISPR enzyme is codon-optimized forexpression in a eukaryotic cell. In some embodiments, the CRISPR enzymedirects cleavage of one or two strands at the location of the targetsequence. In some embodiments, the CRISPR enzyme lacks DNA strandcleavage activity.

In one aspect, the invention provides a CRISPR enzyme comprising one ormore nuclear localization sequences of sufficient strength to driveaccumulation of said CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In some embodiments, the CRISPR enzyme is a typeII CRISPR system enzyme. In some embodiments, the CRISPR enzyme is aCas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S.pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derivedfrom these organisms. The enzyme may be a Cas9 homolog or ortholog. Insome embodiments, the CRISPR enzyme lacks the ability to cleave one ormore strands of a target sequence to which it binds.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a tracr mate sequenceand one or more insertion sites for inserting one or more guidesequences upstream of the tracr mate sequence, wherein when expressed,the guide sequence directs sequence-specific binding of a CRISPR complexto a target sequence in a eukaryotic cell, wherein the CRISPR complexcomprises a CRISPR enzyme complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the tracr mate sequence thatis hybridized to the tracr sequence; and/or (b) a second regulatoryelement operably linked to an enzyme-coding sequence encoding saidCRISPR enzyme comprising a nuclear localization sequence. In someembodiments, the host cell comprises components (a) and (b). In someembodiments, component (a), component (b), or components (a) and (b) arestably integrated into a genome of the host eukaryotic cell. In someembodiments, component (a) further comprises the tracr sequencedownstream of the tracr mate sequence under the control of the firstregulatory element. In some embodiments, component (a) further comprisestwo or more guide sequences operably linked to the first regulatoryelement, wherein when expressed, each of the two or more guide sequencesdirect sequence specific binding of a CRISPR complex to a differenttarget sequence in a eukaryotic cell. In some embodiments, theeukaryotic host cell further comprises a third regulatory element, suchas a polymerase III promoter, operably linked to said tracr sequence. Insome embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,80%, 90%, 95%, or 99% of sequence complementarity along the length ofthe tracr mate sequence when optimally aligned. In some embodiments, theCRISPR enzyme comprises one or more nuclear localization sequences ofsufficient strength to drive accumulation of said CRISPR enzyme in adetectable amount in the nucleus of a eukaryotic cell. In someembodiments, the CRISPR enzyme is a type II CRISPR system enzyme. Insome embodiments, the CRISPR enzyme is a Cas9 enzyme. In someembodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.thermophilus Cas9, and may include mutated Cas9 derived from theseorganisms. The enzyme may be a Cas9 homolog or ortholog. In someembodiments, the CRISPR enzyme is codon-optimized for expression in aeukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavageof one or two strands at the location of the target sequence. In someembodiments, the CRISPR enzyme lacks DNA strand cleavage activity. Insome embodiments, the first regulatory element is a polymerase IIIpromoter. In some embodiments, the second regulatory element is apolymerase 11 promoter. In some embodiments, the guide sequence is atleast 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, orbetween 15-25, or between 15-20 nucleotides in length. In an aspect, theinvention provides a non-human eukaryotic organism; preferably amulticellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. In other aspects, theinvention provides a eukaryotic organism; preferably a multicellulareukaryotic organism, comprising a eukaryotic host cell according to anyof the described embodiments. The organism in some embodiments of theseaspects may be an animal; for example a mammal. Also, the organism maybe an arthropod such as an insect. The organism also may be a plant.Further, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises (a) a first regulatory element operablylinked to a tracr mate sequence and one or more insertion sites forinserting one or more guide sequences upstream of the tracr matesequence, wherein when expressed, the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzymecomplexed with (1) the guide sequence that is hybridized to the targetsequence, and (2) the tracr mate sequence that is hybridized to thetracr sequence; and/or (b) a second regulatory element operably linkedto an enzyme-coding sequence encoding said CRISPR enzyme comprising anuclear localization sequence. In some embodiments, the kit comprisescomponents (a) and (b) located on the same or different vectors of thesystem. In some embodiments, component (a) further comprises the tracrsequence downstream of the tracr mate sequence under the control of thefirst regulatory element. In some embodiments, component (a) furthercomprises two or more guide sequences operably linked to the firstregulatory element, wherein when expressed, each of the two or moreguide sequences direct sequence specific binding of a CRISPR complex toa different target sequence in a eukaryotic cell. In some embodiments,the system further comprises a third regulatory element, such as apolymerase II promoter, operably linked to said tracr sequence. In someembodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%,90%, 95%, or 99% of sequence complementarity along the length of thetracr mate sequence when optimally aligned. In some embodiments, theCRISPR enzyme comprises one or more nuclear localization sequences ofsufficient strength to drive accumulation of said CRISPR enzyme in adetectable amount in the nucleus of a eukaryotic cell. In someembodiments, the CRISPR enzyme is a type II CRISPR system enzyme. Insome embodiments, the CRISPR enzyme is a Cas9 enzyme. In someembodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.thermophilus Cas9, and may include mutated Cas9 derived from theseorganisms. The enzyme may be a Cas9 homolog or ortholog. In someembodiments, the CRISPR enzyme is codon-optimized for expression in aeukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavageof one or two strands at the location of the target sequence. In someembodiments, the CRISPR enzyme lacks DNA strand cleavage activity. Insome embodiments, the first regulatory element is a polymerase IIIpromoter. In some embodiments, the second regulatory element is apolymerase II promoter. In some embodiments, the guide sequence is atleast 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, orbetween 15-25, or between 15-20 nucleotides in length.

In one aspect, the invention provides a method of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.In some embodiments, said cleavage comprises cleaving one or two strandsat the location of the target sequence by said CRISPR enzyme. In someembodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide. In some embodiments,said mutation results in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the method further comprises delivering one or more vectorsto said eukaryotic cell, wherein the one or more vectors driveexpression of one or more of: the CRISPR enzyme, the guide sequencelinked to the tracr mate sequence, and the tracr sequence. In someembodiments, said vectors are delivered to the eukaryotic cell in asubject. In some embodiments, said modifying takes place in saideukaryotic cell in a cell culture. In some embodiments, the methodfurther comprises isolating said eukaryotic cell from a subject prior tosaid modifying. In some embodiments, the method further comprisesreturning said eukaryotic cell and/or cells derived therefrom to saidsubject.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence. Insome embodiments, the method further comprises delivering one or morevectors to said eukaryotic cells, wherein the one or more vectors driveexpression of one or more of: the CRISPR enzyme, the guide sequencelinked to the tracr mate sequence, and the tracr sequence.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated an increase in the risk of havingor developing a disease. In some embodiments, the method comprises (a)introducing one or more vectors into a eukaryotic cell, wherein the oneor more vectors drive expression of one or more of: a CRISPR enzyme, aguide sequence linked to a tracr mate sequence, and a tracr sequence;and (b) allowing a CRISPR complex to bind to a target polynucleotide toeffect cleavage of the target polynucleotide within said disease gene,wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridized to the target sequence withinthe target polynucleotide, and (2) the tracr mate sequence that ishybridized to the tracr sequence, thereby generating a model eukaryoticcell comprising a mutated disease gene. In some embodiments, saidcleavage comprises cleaving one or two strands at the location of thetarget sequence by said CRISPR enzyme. In some embodiments, saidcleavage results in decreased transcription of a target gene. In someembodiments, the method further comprises repairing said cleaved targetpolynucleotide by homologous recombination with an exogenous templatepolynucleotide, wherein said repair results in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of saidtarget polynucleotide. In some embodiments, said mutation results in oneor more amino acid changes in a protein expression from a genecomprising the target sequence.

In one aspect, the invention provides a method for developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. In some embodiments, a disease gene isany gene associated an increase in the risk of having or developing adisease. In some embodiments, the method comprises (a) contacting a testcompound with a model cell of any one of the described embodiments; and(b) detecting a change in a readout that is indicative of a reduction oran augmentation of a cell signaling event associated with said mutationin said disease gene, thereby developing said biologically active agentthat modulates said cell signaling event associated with said diseasegene.

In one aspect, the invention provides a recombinant polynucleotidecomprising a guide sequence upstream of a tracr mate sequence, whereinthe guide sequence when expressed directs sequence-specific binding of aCRISPR complex to a corresponding target sequence present in aeukaryotic cell. In some embodiments, the target sequence is a viralsequence present in a eukaryotic cell. In some embodiments, the targetsequence is a proto-oncogene or an oncogene.

In one aspect the invention provides for a method of selecting one ormore prokaryotic cell(s) by introducing one or more mutations in a genein the one or more prokaryotic cell (s), the method comprising:introducing one or more vectors into the prokaryotic cell (s), whereinthe one or more vectors drive expression of one or more of: a CRISPRenzyme, a guide sequence linked to a tracr mate sequence, a tracrsequence, and a editing template; wherein the editing template comprisesthe one or more mutations that abolish CRISPR enzyme cleavage; allowinghomologous recombination of the editing template with the targetpolynucleotide in the cell(s) to be selected; allowing a CRISPR complexto bind to a target polynucleotide to effect cleavage of the targetpolynucleotide within said gene, wherein the CRISPR complex comprisesthe CRISPR enzyme complexed with (1) the guide sequence that ishybridized to the target sequence within the target polynucleotide, and(2) the tracr mate sequence that is hybridized to the tracr sequence,wherein binding of the CRISPR complex to the target polynucleotideinduces cell death, thereby allowing one or more prokaryotic cell(s) inwhich one or more mutations have been introduced to be selected. In apreferred embodiment, the CRISPR enzyme is Cas9. In another aspect ofthe invention the cell to be selected may be a eukaryotic cell. Aspectsof the invention allow for selection of specific cells without requiringa selection marker or a two-step process that may include acounter-selection system.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”. “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention. These and other embodiments aredisclosed or are obvious from and encompassed by, the following DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a schematic model of the CRISPR system. The Cas9 nucleasefrom Streptococcus pyogenes (yellow) is targeted to genomic DNA by asynthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue)and a scaffold (red). The guide sequence base-pairs with the DNA target(blue), directly upstream of a requisite 5′-NGG protospacer adjacentmotif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ˜3bp upstream of the PAM (red triangle).

FIG. 2A-F shows an exemplary CRISPR system, a possible mechanism ofaction, an example adaptation for expression in eukaryotic cells, andresults of tests assessing nuclear localization and CRISPR activity.FIG. 2C discloses SEQ ID NOS 279-280, respectively, in order ofappearance. FIG. 2E discloses SEQ ID NOS 281-283, respectively, in orderof appearance. FIG. 2F discloses SEQ ID NOS 284-288, respectively, inorder of appearance.

FIG. 3 shows an exemplary expression cassette for expression of CRISPRsystem elements in eukaryotic cells, predicted structures of exampleguide sequences, and CRISPR system activity as measured in eukaryoticand prokaryotic cells (SEQ ID NOS 289-298, respectively, in order ofappearance).

FIG. 4A-D shows results of an evaluation of SpCas9 specificity for anexample target. FIG. 4A discloses SEQ ID NOS 299, 282 and 300-310,respectively, in order of appearance. FIG. 4C discloses SEQ ID NO: 299.

FIG. 5A-G show an exemplary vector system and results for its use indirecting homologous recombination in eukaryotic cells. FIG. 5Ediscloses SEQ ID NO: 311. FIG. 5F discloses SEQ ID NOS 312-313,respectively, in order of appearance. FIG. 5G discloses SEQ ID NOS314-318, respectively, in order of appearance.

FIG. 6 provides a table of protospacer sequences (SEQ ID NOS 33, 32, 31,322-327, 35, 34 and 330-334, respectively, in order of appearance) andsummarizes modification efficiency results for protospacer targetsdesigned based on exemplary S. pyogenes and S. thermophilus CRISPRsystems with corresponding PAMs against loci in human and mouse genomes.Cells were transfected with Cas9 and either pre-crRNA/tracrRNA orchimeric RNA, and analyzed 72 hours after transfection. Percent indelsare calculated based on Surveyor assay results from indicated cell lines(N=3 for all protospacer targets, errors are S.E.M., N.D. indicates notdetectable using the Surveyor assay, and N.T. indicates not tested inthis study).

FIG. 7A-C shows a comparison of different tracrRNA transcripts forCas9-mediated gene targeting. FIG. 7A discloses SEQ ID NOS 335-336,respectively, in order of appearance.

FIG. 8 shows a schematic of a surveyor nuclease assay for detection ofdouble strand break-induced micro-insertions and -deletions.

FIG. 9A-B shows exemplary bicistronic expression vectors for expressionof CRISPR system elements in eukaryotic cells. FIG. 9A discloses SEQ IDNOS 337-339, respectively, in order of appearance. FIG. 9B discloses SEQID NOS 344-342, respectively, in order of appearance.

FIG. 10A-D shows a bacterial plasmid transformation interference assay,expression cassettes and plasmids used therein, and transformationefficiencies of cells used therein. FIG. 10A discloses SEQ ID NOS343-345, respectively, in order of appearance.

FIG. 11A-C shows histograms of distances between adjacent S. pyogenesSF370 locus 1 PAM (NGG) (FIG. 10A) and S. thermophilus LMD9 locus 2 PAM(NNAGAAW) (FIG. 10B) in the human genome; and distances for each PAM bychromosome (Chr) (FIG. 10C).

FIG. 12A-C shows an exemplary CRISPR system, an example adaptation forexpression in eukaryotic cells, and results of tests assessing CRISPRactivity. FIG. 12B discloses SEQ ID NOS 346-347, respectively, in orderof appearance. FIG. 12C discloses SEQ ID NO: 348.

FIG. 13A-C shows exemplary manipulations of a CRISPR system fortargeting of genomic loci in mammalian cells. FIG. 13A discloses SEQ IDNO: 349. FIG. 13B discloses SEQ ID NOS 350-352, respectively, in orderof appearance.

FIG. 14A-B shows the results of a Northern blot analysis of crRNAprocessing in mammalian cells. FIG. 14A discloses SEQ ID NO: 353.

FIG. 15 shows an exemplary selection of protospacers in the human PVALBand mouse Th loci. FIG. 15A discloses SEQ ID NO: 354. FIG. 15B disclosesSEQ ID NO: 355.

FIG. 16 shows example protospacer and corresponding PAM sequence targetsof the S. thermophilus CRISPR system in the human EMX1 locus (SEQ ID NO:348).

FIG. 17 provides a table of sequences for primers and probes (SEQ ID NOS36-39 and 356-363, respectively, in order of appearance) used forSurveyor, RFLP, genomic sequencing, and Northern blot assays.

FIG. 18A-C shows exemplary manipulation of a CRISPR system with chimericRNAs and results of SURVEYOR assays for system activity in eukaryoticcells. FIG. 18A discloses SEQ ID NO: 364, respectively, in order ofappearance.

FIG. 19A-B shows a graphical representation of the results of SURVEYORassays for CRISPR system activity in eukaryotic cells (SEQ ID NOS365-443, respectively, in order of appearance).

FIG. 20 shows an exemplary visualization of some S. pyogenes Cas9 targetsites in the human genome using the UCSC genome browser.

FIG. 21 shows predicted secondary structures for exemplary chimeric RNAscomprising a guide sequence, tracr mate sequence, and tracr sequence(SEQ ID NOS 444-463, respectively, in order of appearance).

FIG. 22 shows exemplary bicistronic expression vectors for expression ofCRISPR system elements in eukaryotic cells (SEQ ID NOS 464 and 341-342,respectively, in order of appearance).

FIG. 23A-B shows that Cas9 nuclease activity against endogenous targetsmay be exploited for genome editing. (a) Concept of genome editing usingthe CRISPR system. The CRISPR targeting construct directed cleavage of achromosomal locus and was co-transformed with an editing template thatrecombined with the target to prevent cleavage. Kanamycin-resistanttransformants that survived CRISPR attack contained modificationsintroduced by the editing template. tracr, trans-activating CRISPR RNA;aphA-3, kanamycin resistance gene. (b) Transformation of crR6M DNA inR6^(8232.5) cells with no editing template, the R6 wild-type srtA or theR6370.1 editing templates. Recombination of either R6 srtA or R6^(370.1)prevented cleavage by Cas9. Transformation efficiency was calculated ascolony forming units (cfu) per μg of crR6M DNA; the mean values withstandard deviations from at least three independent experiments areshown. PCR analysis was performed on 8 clones in each transformation.“Un.” indicates the unedited srtA locus of strain R6^(8232.5); “Ed.”shows the editing template. R6^(8232.5) and R6^(370.1) targets aredistinguished by restriction with EaeI.

FIG. 24A-C shows analysis of PAM and seed sequences that eliminate Cas9cleavage. (a) PCR products with randomized PAM sequences or randomizedseed sequences were transformed in crR6 cells (SEQ ID NOS 465-469,respectively, in order of appearance). These cells expressed Cas9 loadedwith a crRNA that targeted a chromosomal region of R6^(8232.5) cells(highlighted in pink) that is absent from the R6 genome. More than 2×105chloramphenicol-resistant transformants, carrying inactive PAM or seedsequences, were combined for amplification and deep sequencing of thetarget region. (b) Relative proportion of number of reads aftertransformation of the random PAM constructs in crR6 cells (compared tonumber of reads in R6 transformants). The relative abundance for each3-nucleotide PAM sequence is shown. Severely underrepresented sequences(NGG) are shown in red; partially underrepresented one in orange (NAG)(c) Relative proportion of number of reads after transformation of therandom seed sequence constructs in crR6 cells (compared to number ofreads in R6 transformants). The relative abundance of each nucleotidefor each position of the first 20 nucleotides of the protospacersequence is shown (SEQ ID NO: 470). High abundance indicates lack ofcleavage by Cas9, i.e. a CRISPR inactivating mutation. The grey lineshows the level of the WT sequence. The dotted line represents the levelabove which a mutation significantly disrupts cleavage (See section“Analysis of deep sequencing data” in Example 5)

FIG. 25A-F shows introduction of single and multiple mutations using theCRISPR system in S. pneumoniae. (a) Nucleotide and amino acid sequencesof the wild-type and edited (green nucleotides; underlined amino acidresidues) bgaA. The protospacer, PAM and restriction sites are shown(SEQ ID NOS 471-475 and 472, respectively, in order of appearance). (b)Transformation efficiency of cells transformed with targeting constructsin the presence of an editing template or control. (c) PCR analysis for8 transformants of each editing experiment followed by digestion withBtgZI (R→A) and TseI (NE→AA). Deletion of bgaA was revealed as a smallerPCR product. (d) Miller assay to measure the β-galactosidase activity ofWT and edited strains. (e) For a single-step, double deletion thetargeting construct contained two spacers (in this case matching srtAand bgaA) and was co-transformed with two different editing templates(f) PCR analysis for 8 transformants to detect deletions in srtA andbgaA loci. 6/8 transformants contained deletions of both genes.

FIG. 26A-D provides mechanisms underlying editing using the CRISPRsystem. (a) A stop codon was introduced in the erythromycin resistancegene ermAM to generate strain JEN53. The wild-type sequence can berestored by targeting the stop codon with the CRISPR::ermAM(stop)construct, and using the ermAM wild-type sequence as an editingtemplate. (b) Mutant and wild-type ermAM sequences (SEQ ID NOS 476-479,respectively, in order of appearance). (c) Fraction oferythromicyn-resistant (erm^(R)) cfu calculated from total orkanamycin-resistant (kan^(R)) cfu. (d) Fraction of total cells thatacquire both the CRISPR construct and the editing template.Co-transformation of the CRISPR targeting construct produced moretransformants (t-test, p=0.011). In all cases the values show themean±s.d. for three independent experiments.

FIG. 27A-D illustrates genome editing with the CRISPR system in E. coli.(a) A kanamycin-resistant plasmid carrying the CRISPR array (pCRISPR)targeting the gene to edit may be transformed in the HME63recombineering strain containing a chloramphenicol-resistant plasmidharboring cas9 and tracr (pCas9), together with an oligonucleotidespecifying the mutation. (b) A K42T mutation conferring streptomycinresistance was introduced in the rpsL gene (SEQ ID NOS 480-483,respectively, in order of appearance) (c) Fraction ofstreptomicyn-resistant (strep^(R)) cfu calculated from total orkanamycin-resistant (kan^(R)) cfu. (d) Fraction of total cells thatacquire both the pCRISPR plasmid and the editing oligonucleotide.Co-transformation of the pCRISPR targeting plasmid produced moretransformants (t-test, p=0.004). In all cases the values showed themean±s.d. for three independent experiments.

FIG. 28A-B illustrates the transformation of crR6 genomic DNA leads toediting of the targeted locus (a) The IS1167 element of S. pneumoniae R6was replaced by the CRISPR01 locus of S. pyogenes SF370 to generate crR6strain. This locus encodes for the Cas9 nuclease, a CRISPR array withsix spacers, the tracrRNA that is required for crRNA biogenesis andCas1, Cas2 and Csn2, proteins not necessary for targeting. Strain crR6Mcontains a minimal functional CRISPR system without cas1, cas2 and csn2.The aphA-3 gene encodes kanamycin resistance. Protospacers from thestreptococcal bacteriophages φ8232.5 and φ370.1 were fused to achloramphenicol resistance gene (cat) and integrated in the sriA gene ofstrain R6 to generate strains R68232.5 and R6370.1. (b) Left panel:Transformation of crR6 and crR6M genomic DNA in R6^(8232.5) andR^(6370.1). As a control of cell competence a streptomycin resistantgene was also transformed. Right panel: PCR analysis of 8 R6^(8232.5)transformants with crR6 genomic DNA. Primers that amplify the srtA locuswere used for PCR. 7/8 genotyped colonies replaced the R68232.5 srtAlocus by the WT locus from the crR6 genomic DNA.

FIG. 29A-F provides chromatograms of DNA sequences of edited cellsobtained in this study. In all cases the wild-type and mutantprotospacer and PAM sequences (or their reverse complement) areindicated. When relevant, the amino acid sequence encoded by theprotospacer is provided. For each editing experiment, all strains forwhich PCR and restriction analysis corroborated the introduction of thedesired modification were sequenced. A representative chromatogram isshown. (a) Chromatogram for the introduction of a PAM mutation into theR6^(8232.5) target (FIG. 23d ) (SEQ ID NOS 484-485, respectively, inorder of appearance). (b) Chromatograms for the introduction of the R>Aand NE>AA mutations into β-galactosidase (bgaS) (FIG. 25c ) (SEQ ID NOS471-475 and 472, respectively, in order of appearance). (c) Chromatogramfor the introduction of a 6664 bp deletion within bgaA ORF (FIGS. 25cand 25f ). The dotted line indicates the limits of the deletion (SEQ IDNOS 486-488, respectively, in order of appearance). (d) Chromatogram forthe introduction of a 729 bp deletion within srtA ORF (FIG. 25f ). Thedotted line indicates the limits of the deletion (SEQ ID NOS 489-491,respectively, in order of appearance). (e) Chromatograms for thegeneration of a premature stop codon within ermAM (FIG. 33) (SEQ ID NOS492-495, respectively, in order of appearance). (f) rpsL editing in E.coli (FIG. 27) (SEQ ID NOS 480-483, respectively, in order ofappearance).

FIG. 30A-C illustrates CRISPR immunity against random S. pneumoniaetargets containing different PAMs. (a) Position of the 10 random targetson the S. pneumoniae R6 genome. The chosen targets have different PAMsand are on both strands. (b) Spacers corresponding to the targets werecloned in a minimal CRISPR array on plasmid pLZ12 and transformed intostrain crR6Rc, which supplies the processing and targeting machinery intrans. (c) Transformation efficiency of the different plasmids in strainR6 and crR6Rc. No colonies were recovered for the transformation ofpDB99-108 (T1-T10) in crR6Rc. The dashed line represents limit ofdetection of the assay.

FIG. 31 provides a general scheme for targeted genome editing. Tofacilitate targeted genome editing, crR6M was further engineered tocontain tracrRNA. Cas9 and only one repeat of the CRISPR array followedby kanamycin resistance marker (aphA-3), generating strain crR6Rk. DNAfrom this strain is used as a template for PCR with primers designed tointroduce a new spacer (green box designated with N). The left and rightPCRs are assembled using the Gibson method to create the targetingconstruct. Both the targeting and editing constructs are thentransformed into strain crR6Rc, which is a strain equivalent to crR6Rkbut has the kanamycin resistance marker replaced by a chloramphenicolresistance marker (cat). About 90% of the kanamycin-resistanttransformants contain the desired mutation.

FIG. 32 illustrates the distribution of distances between PAMs. NGG andCCN that are considered to be valid PAMs. Data is shown for the S.pneumoniae R6 genome as well as for a random sequence of the same lengthand with the same GC-content (39.7%). The dotted line represents theaverage distance (12) between PAMs in the R6 genome.

FIG. 33A-D illustrates CRISPR-mediated editing of the ermAM locus usinggenomic DNA as targeting construct. To use genomic DNA as targetingconstruct it is necessary to avoid CRISPR autoimmunity, and therefore aspacer against a sequence not present in the chromosome must be used (inthis case the ermAM erythromycin resistance gene). (a) Nucleotide andamino acid sequences of the wild-type and mutated (red letters) ermAMgene. The protospacer and PAM sequences are shown (SEQ ID NOS 492-495,respectively, in order of appearance). (b) A schematic forCRISPR-mediated editing of the ermAM locus using genomic DNA. Aconstruct carrying an ermAM-targeting spacer (blue box) is made by PCRand Gibson assembly, and transformed into strain crR6Rc, generatingstrain JEN37. The genomic DNA of JEN37 was then used as a targetingconstruct, and was co-transformed with the editing template into JEN38,a strain in which the srtA gene was replaced by a wild-type copy ofermAM. Kanamycin-resistant transformants contain the edited genotype(JEN43). (c) Number of kanamycin-resistant cells obtained afterco-transformation of targeting and editing or control templates. In thepresence of the control template 5.4×10³ cfu/ml were obtained, and4.3×10⁵ cfu/ml when the editing template was used. This differenceindicates an editing efficiency of about 99%[(4.3×10⁵-5.4×10³)/4.3×10⁵]. (d) To check for the presence of editedcells seven kanamycin-resistant clones and JEN38 were streaked on agarplates with (erm+) or without (erm−) erythromycin. Only the positivecontrol displayed resistance to erythromycin. The ermAM mut genotype ofone of these transformants was also verified by DNA sequencing (FIG. 29e).

FIG. 34A-D illustrates sequential introduction of mutations byCRISPR-mediated genome editing. (a) A schematic for sequentialintroduction of mutations by CRISPR-mediated genome editing. First, R6is engineered to generate crR6Rk. crR6Rk is co-transformed with asrtA-targeting construct fused to cat for chloramphenicol selection ofedited cells, along with an editing construct for a ΔsrtA in-framedeletion. Strain crR6 ΔsrtA is generated by selection on chlramphenicol.Subsequently, the ΔsrtA strain is co-transformed with a bgaA-targetingconstruct fused to aphA-3 for kanamycin selection of edited cells, andan editing construct containing a ΔbgaA in-frame deletion. Finally, theengineered CRISPR locus can be erased from the chromosome by firstco-transforming R6 DNA containing the wild-type IS1167 locus and aplasmid carrying a bgaA protospacer (pDB97), and selection onspectinomycin. (b) PCR analysis for 8 chloramphenicol (Cam)-resistanttransformants to detect the deletion in the srtA locus. (c)β-galactosidase activity as measured by Miller assay. In S. pneumoniae,this enzyme is anchored to the cell wall by sortase A. Deletion of thesrtA gene results in the release of β-galactosidase into thesupernatant. ΔbgaA mutants show no activity. (d) PCR analysis for 8spectinomycin (Spec)-resistant transformants to detect the replacementof the CRISPR locus by wild-type IS1167.

FIG. 35A-C illustrates the background mutation frequency of CRISPR in S.pneumoniae. (a) Transformation of the CRISPR::Ø or CRISPR::erm(stop)targeting constructs in JEN53, with or without the ermAM editingtemplate. The difference in kan^(R) CFU between CRISPR::Ø andCRISPR::erm(stop) indicates that Cas9 cleavage kills non-edited cells.Mutants that escape CRISPR interference in the absence of editingtemplate are observed at a frequency of 3×10⁻³. (b) PCR analysis of theCRISPR locus of escapers shows that 7/8 have a spacer deletion. (c)Escaper #2 carries a point mutation in cas9 (SEQ ID NOS 496-499,respectively, in order of appearance).

FIG. 36 illustrates that the essential elements of the S. pyogenesCRISPR locus 1 are reconstituted in E. coli using pCas9. The plasmidcontained tracrRNA, Cas9, as well as a leader sequence driving the crRNAarray. The pCRISPR plasmids contained the leader and the array only.Spacers may be inserted into the crRNA array between BsaI sites usingannealed oligonucleotides (SEQ ID NOS 343, 500 and 127, respectively, inorder of appearance). Oligonucleotide design is shown at bottom. pCas9carried chloramphenicol resistance (CmR) and is based on the low-copypACYC184 plasmid backbone. pCRISPR is based on the high-copy numberpZE21 plasmid. Two plasmids were required because a pCRISPR plasmidcontaining a spacer targeting the E. coli chromosome may not beconstructed using this organism as a cloning host if Cas9 is alsopresent (it will kill the host).

FIG. 37 illustrates CRISPR-directed editing in E. coli MG1655. Anoligonucleotide (W542) carrying a point mutation that both confersstreptomycin resistance and abolishes CRISPR immunity, together with aplasmid targeting rpsL (pCRISPR::rpsL) or a control plasmid (pCRISPR::Ø)were co-transformed into wild-type E. coli strain MG1655 containingpCas9. Transformants were selected on media containing eitherstreptomycin or kanamycin. Dashed line indicates limit of detection ofthe transformation assay.

FIG. 38A-B illustrates the background mutation frequency of CRISPR in E.coli HME63. (a) Transformation of the pCRISPR::Ø or pCRISPR::rpsLplasmids into HME63 competent cells. Mutants that escape CRISPRinterference were observed at a frequency of 2.6×10⁻⁴. (b) Amplificationof the CRISPR array of escapers showed that 8/8 have deleted the spacer.

FIG. 39A-D shows a circular depiction of the phylogenetic analysisrevealing five families of Cas9s, including three groups of large Cas9s(˜1400 amino acids) and two of small Cas9s (˜1100 amino acids).

FIG. 40A-F shows the linear depiction of the phylogenetic analysisrevealing five families of Cas9s, including three groups of large Cas9s(˜1400 amino acids) and two of small Cas9s (˜1100 amino acids).

FIG. 41A-M shows sequences where the mutation points are located withinthe SpCas9 gene (SEQ ID NOS 501-502, respectively, in order ofappearance).

FIG. 42 shows a schematic construct in which the transcriptionalactivation domain (VP64) is fused to Cas9 with two mutations in thecatalytic domains (D10 and H840).

FIG. 43A-D shows genome editing via homologous recombination. (a)Schematic of SpCas9 nickase, with D10A mutation in the RuvC I catalyticdomain. (b) Schematic representing homologous recombination (HR) at thehuman EMX1 locus using either sense or antisense single strandedoligonucleotides as repair templates. Red arrow above indicates sgRNAcleavage site; PCR primers for genotyping (Tables J and K) are indicatedas arrows in right panel. (c) Sequence of region modified by HR. d,SURVEYOR assay for wildtype (wt) and nickase (D10A) SpCas9-mediatedindels at the EMX1 target 1 locus (n=3) (SEQ ID NOS 503-505, 503, 506and 505, respectively, in order of appearance). Arrows indicatepositions of expected fragment sizes.

FIG. 44A-B shows single vector designs for SpCas9. FIG. 44A disclosesSEQ ID NOS 320-321 and 328, respectively, in order of appearance. FIG.44B discloses SEQ ID NO: 329.

FIG. 45 shows quantification of cleavage of NLS-Csn1 constructsNLS-Csn1, Csn1, Csn1-NLS, NLS-Csn1-NLS, NLS-Csn1-GFP-NLS and UnTFN.

FIG. 46 shows index frequency of NLS-Cas9, Cas9, Cas9-NLS andNLS-Cas9-NLS.

FIG. 47 shows a gel demonstrating that SpCas9 with nickase mutations(individually) do not induce double strand breaks.

FIG. 48A-B shows a design of the oligo DNA used as HomologousRecombination (HR) template in this experiment and a comparison of HRefficiency induced by different combinations of Cas9 protein and HRtemplate.

FIG. 49A shows the Conditional Cas9, Rosa26 targeting vector map.

FIG. 49B shows the Constitutive Cas9, Rosa26 targeting vector map.

FIG. 50A-H show the sequences of each element present in the vector mapsof FIGS. 49A-B (SEQ ID NOS 507-516, respectively, in order ofappearance).

FIG. 51 shows a schematic of the important elements in the Constitutiveand Conditional Cas9 constructs.

FIG. 52 shows the functional validation of the expression ofConstitutive and Conditional Cas9 constructs.

FIG. 53 shows the validation of Cas9 nuclease activity by Surveyor.

FIG. 54 shows the quantification of Cas9 nuclease activity.

FIG. 55 shows construct design and homologous recombination (HR)strategy.

FIG. 56 shows the genomic PCR genotyping results for the constitutive(Right) and conditional (Left) constructs at two different gel exposuretimes (top row for 3 min and bottom row for 1 min).

FIG. 57 shows Cas9 activation in mESCs.

FIG. 58 shows a schematic of the strategy used to mediate gene knockoutvia NHEJ using a nickase version of Cas9 along with two guide RNAs.

FIG. 59 shows how DNA double-strand break (DSB) repair promotes geneediting. In the error-prone non-homologous end joining (NHEJ) pathway,the ends of a DSB are processed by endogenous DNA repair machineries andrejoined together, which can result in random insertion/deletion (indel)mutations at the site of junction. Indel mutations occurring within thecoding region of a gene can result in frame-shift and a premature stopcodon, leading to gene knockout. Alternatively, a repair template in theform of a plasmid or single-stranded oligodeoxynucleotides (ssODN) canbe supplied to leverage the homology-directed repair (HDR) pathway,which allows high fidelity and precise editing.

FIG. 60 shows the timeline and overview of experiments. Steps forreagent design, construction, validation, and cell line expansion.Custom sgRNAs (light blue bars) for each target, as well as genotypingprimers, are designed in silico via our online design tool (available atthe website genome-engineering.org/tools). sgRNA expression vectors arethen cloned into a plasmid containing Cas9 (PX330) and verified via DNAsequencing. Completed plasmids (pCRISPRs), and optional repair templatesfor facilitating homology directed repair, are then transfected intocells and assayed for ability to mediate targeted cleavage. Finally,transfected cells can be clonally expanded to derive isogenic cell lineswith defined mutations.

FIG. 61A-C shows Target selection and reagent preparation. (a) For S.pyogenes Cas9, 20-bp targets (highlighted in blue) must be followed by5′-NGG, which can occur in either strand on genomic DNA. We recommendusing the online tool described in this protocol in aiding targetselection (www.genome-engineering.org/tools). (b) Schematic forco-transfection of Cas9 expression plasmid (PX165) and PCR-amplifiedU6-driven sgRNA expression cassette. Using a U6 promoter-containing PCRtemplate and a fixed forward primer (U6 Fwd), sgRNA-encoding DNA canappended onto the U6 reverse primer (U6 Rev) and synthesized as anextended DNA oligo (Ultramer oligos from IDT). Note the guide sequence(blue N's) in U6 Rev is the reverse complement of the 5′-NGG flankingtarget sequence (SEQ ID NOS 517 and 517-519, respectively, in order ofappearance). (c) Schematic for scarless cloning of the guide sequenceoligos into a plasmid containing Cas9 and sgRNA scaffold (PX330). Theguide oligos (blue N's) contain overhangs for ligation into the pair ofBbsI sites on PS330, with the top and bottom strand orientationsmatching those of the genomic target (i.e. top oligo is the 20-bpsequence preceding 5′-NGG in genomic DNA). Digestion of PX330 with BbsIallows the replacement of the Type IIs restriction sites (blue outline)with direct insertion of annealed oligos. It is worth noting that anextra G was placed before the first base of the guide sequence.Applicants have found that an extra G in front of the guide sequencedoes not adversely affect targeting efficiency. In cases when the 20-ntguide sequence of choice does not begin with guanine, the extra guaninewill ensure the sgRNA is efficiently transcribed by the U6 promoter,which prefers a guanine in the first base of the transcript (SEQ ID NOS320-321 and 328, respectively, in order of appearance).

FIG. 62A-D shows the anticipated results for multiplex NHEJ. (a)Schematic of the SURVEYOR assay used to determine indel percentage.First, genomic DNA from the heterogeneous population of Cas9-targetedcells is amplified by PCR. Amplicons are then reannealed slowly togenerate heteroduplexes. The reannealed heteroduplexes are cleaved bySURVEYOR nuclease, whereas homoduplexes are left intact. Cas9-mediatedcleavage efficiency (% indel) is calculated based on the fraction ofcleaved DNA, as determined by integrated intensity of gel bands. (b) TwosgRNAs (orange and blue bars) are designed to target the human GRIN2Band DYRK1A loci. SURVEYOR gel shows modification at both loci intransfected cells. Colored arrows indicated expected fragment sizes foreach locus. (c) A pair of sgRNAs (light blue and green bars) aredesigned to excise an exon (dark blue) in the human EMX1 locus. Targetsequences and PAMs (red) are shown in respective colors, and sites ofcleavage indicated by red triangle. Predicted junction is shown below.Individual clones isolated from cell populations transfected with sgRNA3, 4, or both are assayed by PCR (OUT Fwd, OUT Rev), reflecting adeletion of ˜270-bp. Representative clones with no modification (12/23),mono-allelic (10/23), and bi-allelic (1/23) modifications are shown. INFwd and IN Rev primers are used to screen for inversion events (FIG. 6d) (SEQ ID NOS 520-522, respectively, in order of appearance). (d)Quantification of clonal lines with EMX1 exon deletions. Two pairs ofsgRNAs (3.1, 3.2 left-flanking sgRNAs; 4.1, 4.2, right flanking sgRNAs)are used to mediate deletions of variable sizes around one EMX1 exon.Transfected cells are clonally isolated and expanded for genotypinganalysis for deletions and inversion events. Of the 105 clones arescreened, 51 (49%) and 11 (10%) carrying heterozygous and homozygousdeletions, respectively. Approximate deletion sizes are given sincejunctions may be variable.

FIG. 63A-C shows the application of ssODNs and targeting vector tomediate HR with both wildtype and nickase mutant of Cas9 in HEK293FT andHUES9 cells with efficiencies ranging from 1.0-27%. FIG. 63B disclosesSEQ ID NOS 503-505, 503, 506 and 505, respectively, in order ofappearance.

FIG. 64 shows a schematic of a PCR-based method for rapid and efficientCRISPR targeting in mammalian cells. A plasmid containing the human RNApolymerase III promoter U6 is PCR-amplified using a U6-specific forwardprimer and a reverse primer carrying the reverse complement of part ofthe U6 promoter, the sgRNA(+85) scaffold with guide sequence, and 7 Tnucleotides for transcriptional termination. The resulting PCR productis purified and co-delivered with a plasmid carrying Cas9 driven by theCBh promoter (SEQ ID NOS 517, 523, 518 and 524-525, respectively, inorder of appearance).

FIG. 65 shows SURVEYOR Mutation Detection Kit from Transgenomics resultsfor each gRNA and respective controls. A positive SURVEYOR result is onelarge band corresponding to the genomic PCR and two smaller bands thatare the product of the SURVEYOR nuclease making a double-strand break atthe site of a mutation. Each gRNA was validated in the mouse cell line.Neuro-N2a, by liposomal transient co-transfection with hSpCas9. 72 hourspost-transfection genomic DNA was purified using QuickExtract DNA fromEpicentre. PCR was performed to amplify the locus of interest.

FIG. 66 shows Surveyor results for 38 live pups (lanes 1-38) 1 dead pup(lane 39) and 1 wild-type pup for comparison (lane 40). Pups 1-19 wereinjected with gRNA Chd8.2 and pups 20-38 were injected with gRNA Chd8.3.Of the 38 live pups, 13 were positive for a mutation. The one dead pupalso had a mutation. There was no mutation detected in the wild-typesample. Genomic PCR sequencing was consistent with the SURVEYOR assayfindings (SEQ ID NOS 526-528, respectively, in order of appearance).

FIG. 67 shows a design of different Cas9 NLS constructs. All Cas9 werethe human-codon-optimized version of the Sp Cas9. NLS sequences arelinked to the cas9 gene at either N-terminus or C-terminus. All Cas9variants with different NLS designs were cloned into a backbone vectorcontaining so it is driven by EF1a promoter. On the same vector there isa chimeric RNA targeting human EMX1 locus driven by U6 promoter,together forming a two-component system.

FIG. 68 shows the efficiency of genomic cleavage induced by Cas9variants bearing different NLS designs. The percentage indicate theportion of human EMX1 genomic DNA that were cleaved by each construct.All experiments are from 3 biological replicates. n=3, error indicatesS.E.M.

FIG. 69A shows a design of the CRISPR-TF (Transcription Factor) withtranscriptional activation activity. The chimeric RNA is expressed by U6promoter, while a human-codon-optimized, double-mutant version of theCas9 protein (hSpCas9m), operably linked to triple NLS and a VP64functional domain is expressed by a EF1a promoter. The double mutations,D10A and H840A, renders the cas9 protein unable to introduce anycleavage but maintained its capacity to bind to target DNA when guidedby the chimeric RNA.

FIG. 69B shows transcriptional activation of the human SOX2 gene withCRISPR-TF system (Chimeric RNA and the Cas9-NLS-VP64 fusion protein).293FT cells were transfected with plasmids bearing two components: (1)U6-driven different chimeric RNAs targeting 20-bp sequences within oraround the human SOX2 genomic locus, and (2) EF1a-driven hSpCas9m(double mutant)-NLS-VP64 fusion protein. 96 hours post transfection,293FT cells were harvested and the level of activation is measured bythe induction of mRNA expression using a qRT-PCR assay. All expressionlevels are normalized against the control group (grey bar), whichrepresents results from cells transfected with the CRISPR-TF backboneplasmid without chimeric RNA. The qRT-PCR probes used for detecting theSOX2 mRNA is Taqman Human Gene Expression Assay (Life Technologies). Allexperiments represents data from 3 biological replicates, n=3, errorbars show s.e.m.

FIG. 70 depicts NLS architecture optimization for SpCas9.

FIG. 71 shows a QQ plot for NGGNN sequences.

FIG. 72 shows a histogram of the data density with fitted normaldistribution (black line) and 0.99 quantile (dotted line).

FIG. 73A-C shows RNA-guided repression of bgaA expression bydgRNA::cas9**. a. The Cas9 protein binds to the tracrRNA, and to theprecursor CRISPR RNA which is processed by RNAseIII to form the crRNA.The crRNA directs binding of Cas9 to the bgaA promoter and repressestranscription. b. The targets used to direct Cas9** to the bgaA promoterare represented (SEQ ID NO: 529). Putative −35, −10 as well as the bgaAstart codon are in bold. c. Betagalactosidase activity as measure byMiller assay in the absence of targeting and for the four differenttargets.

FIG. 74A-E shows characterization of Cas9** mediated repression. a. Theg/jmut2 gene and its promoter, including the −35 and −10 signals arerepresented together with the position of the different target sitesused the study. b. Relative fluorescence upon targeting of the codingstrand. c. Relative fluorescence upon targeting of the non-codingstrand. d. Northern blot with probes B477 and B478 on RNA extracted fromT5, T10, B10 or a control strain without a target. e. Effect of anincreased number of mutations in the 5′ end of the crRNA of B1, T5 andB10.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide maycomprise one or more modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.

In aspects of the invention the terms “chimeric RNA”, “chimeric guideRNA”, “guide RNA”, “single guide RNA” and “synthetic guide RNA” are usedinterchangeably and refer to the polynucleotide sequence comprising theguide sequence, the tracr sequence and the tracr mate sequence. The term“guide sequence” refers to the about 20 bp sequence within the guide RNAthat specifies the target site and may be used interchangeably with theterms “guide” or “spacer”. The term “tracr mate sequence” may also beused interchangeably with the term “direct repeat(s)”.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick base pairing or other non-traditional types. Apercent complementarity indicates the percentage of residues in anucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% over a region of 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology-Hybridization With Nucleic Acid Probes Part I, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Vectors may be introduced and propagated in a prokaryote. In someembodiments, a prokaryote is used to amplify copies of a vector to beintroduced into a eukaryotic cell or as an intermediate vector in theproduction of a vector to be introduced into a eukaryotic cell (e.g.amplifying a plasmid as part of a viral vector packaging system). Insome embodiments, a prokaryote is used to amplify copies of a vector andexpress one or more nucleic acids, such as to provide a source of one ormore proteins for delivery to a host cell or host organism. Expressionof proteins in prokaryotes is most often carried out in Escherichia coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, such as to theamino terminus of the recombinant protein. Such fusion vectors may serveone or more purposes, such as: (i) to increase expression of recombinantprotein; (ii) to increase the solubility of the recombinant protein; and(iii) to aid in the purification of the recombinant protein by acting asa ligand in affinity purification. Often, in fusion expression vectors,a proteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET IId(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerivisae include pYepSec1(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277). lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “CRISPR system” refers collectively to transcripts and otherelements involved in the expression of or directing the activity ofCRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus. In some embodiments, oneor more elements of a CRISPR system is derived from a type 1, type II,or type III CRISPR system. In some embodiments, one or more elements ofa CRISPR system is derived from a particular organism comprising anendogenous CRISPR system, such as Streptococcus pyogenes. In general, aCRISPR system is characterized by elements that promote the formation ofa CRISPR complex at the site of a target sequence (also referred to as aprotospacer in the context of an endogenous CRISPR system). In thecontext of formation of a CRISPR complex, “target sequence” refers to asequence to which a guide sequence is designed to have complementarity,where hybridization between a target sequence and a guide sequencepromotes the formation of a CRISPR complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a CRISPR complex. A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, the targetsequence may be within an organelle of a eukaryotic cell, for example,mitochondrion or chloroplast. A sequence or template that may be usedfor recombination into the targeted locus comprising the targetsequences is referred to as an “editing template” or “editingpolynucleotide” or “editing sequence”. In aspects of the invention, anexogenous template polynucleotide may be referred to as an editingtemplate. In an aspect of the invention the recombination is homologousrecombination.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.Without wishing to be bound by theory, the tract sequence, which maycomprise or consist of all or a portion of a wild-type tracr sequence(e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, ormore nucleotides of a wild-type tracr sequence), may also form part of aCRISPR complex, such as by hybridization along at least a portion of thetracr sequence to all or a portion of a tracr mate sequence that isoperably linked to the guide sequence. In some embodiments, the tracrsequence has sufficient complementarity to a tract mate sequence tohybridize and participate in formation of a CRISPR complex. As with thetarget sequence, it is believed that complete complementarity is notneeded, provided there is sufficient to be functional. In someembodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%,95% or 99% of sequence complementarity along the length of the tracrmate sequence when optimally aligned. In some embodiments, one or morevectors driving expression of one or more elements of a CRISPR systemare introduced into a host cell such that expression of the elements ofthe CRISPR system direct formation of a CRISPR complex at one or moretarget sites. For example, a Cas enzyme, a guide sequence linked to atracr-mate sequence, and a tracr sequence could each be operably linkedto separate regulatory elements on separate vectors. Alternatively, twoor more of the elements expressed from the same or different regulatoryelements, may be combined in a single vector, with one or moreadditional vectors providing any components of the CRISPR system notincluded in the first vector. CRISPR system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a CRISPR enzyme and one or more of the guidesequence, tracr mate sequence (optionally operably linked to the guidesequence), and a tracr sequence embedded within one or more intronsequences (e.g. each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the CRISPRenzyme, guide sequence, tracr mate sequence, and tracr sequence areoperably linked to and expressed from the same promoter.

In some embodiments, a vector comprises one or more insertion sites,such as a restriction endonuclease recognition sequence (also referredto as a “cloning site”). In some embodiments, one or more insertionsites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore insertion sites) are located upstream and/or downstream of one ormore sequence elements of one or more vectors. In some embodiments, avector comprises an insertion site upstream of a tracr mate sequence,and optionally downstream of a regulatory element operably linked to thetracr mate sequence, such that following insertion of a guide sequenceinto the insertion site and upon expression the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell. In some embodiments, a vector comprises two or moreinsertion sites, each insertion site being located between two tracrmate sequences so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences maycomprise two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target CRISPR activity to multiple different, correspondingtarget sequences within a cell. For example, a single vector maycomprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or more guide sequences. In some embodiments, about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containingvectors may be provided, and optionally delivered to a cell.

In some embodiments, a vector comprises a regulatory element operablylinked to an enzyme-coding sequence encoding a CRISPR enzyme, such as aCas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, homologs thereof, or modified versions thereof. Theseenzymes are known; for example, the amino acid sequence of S. pyogenesCas9 protein may be found in the SwissProt database under accessionnumber Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNAcleavage activity, such as Cas9. In some embodiments the CRISPR enzymeis Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. In someembodiments, the CRISPR enzyme directs cleavage of one or both strandsat the location of a target sequence, such as within the target sequenceand/or within the complement of the target sequence. In someembodiments, the CRISPR enzyme directs cleavage of one or both strandswithin about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200,500, or more base pairs from the first or last nucleotide of a targetsequence. In some embodiments, a vector encodes a CRISPR enzyme that ismutated to with respect to a corresponding wild-type enzyme such thatthe mutated CRISPR enzyme lacks the ability to cleave one or bothstrands of a target polynucleotide containing a target sequence. Forexample, an aspartate-to-alanine substitution (D10A) in the RuvC Icatalytic domain of Cas9 from S. pyogenes converts Cas9 from a nucleasethat cleaves both strands to a nickase (cleaves a single strand). Otherexamples of mutations that render Cas9 a nickase include, withoutlimitation, H840A, N854A, and N863A. In some embodiments, a Cas9 nickasemay be used in combination with guide sequenc(es), e.g., two guidesequences, which target respectively sense and antisense strands of theDNA target. This combination allows both strands to be nicked and usedto induce NHEJ. Applicants have demonstrated (data not shown) theefficacy of two nickase targets (i.e., sgRNAs targeted at the samelocation but to different strands of DNA) in inducing mutagenic NHEJ. Asingle nickase (Cas9-D10A with a single sgRNA) is unable to induce NHEJand create indels but Applicants have shown that double nickase(Cas9-D10A and two sgRNAs targeted to different strands at the samelocation) can do so in human embryonic stem cells (hESCs). Theefficiency is about 50% of nuclease (i.e., regular Cas9 without D10mutation) in hESCs.

As a further example, two or more catalytic domains of Cas9 (RuvC I,RuvC II, and RuvC III) may be mutated to produce a mutated Cas9substantially lacking all DNA cleavage activity. In some embodiments, aD10A mutation is combined with one or more of H840A, N854A, or N863Amutations to produce a Cas9 enzyme substantially lacking all DNAcleavage activity. In some embodiments, a CRISPR enzyme is considered tosubstantially lack all DNA cleavage activity when the DNA cleavageactivity of the mutated enzyme is less than about 25%, 10%, 5%, 1%,0.1%, 0.01%, or lower with respect to its non-mutated form. Othermutations may be useful; where the Cas9 or other CRISPR enzyme is from aspecies other than S. pyogenes, mutations in corresponding amino acidsmay be made to achieve similar effects.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzymeis codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, mouse, rat, rabbit, dog, or non-human primate. In general, codonoptimization refers to a process of modifying a nucleic acid sequencefor enhanced expression in the host cells of interest by replacing atleast one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15,20, 25, 50, or more codons) of the native sequence with codons that aremore frequently or most frequently used in the genes of that host cellwhile maintaining the native amino acid sequence. Various speciesexhibit particular bias for certain codons of a particular amino acid.Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database”, and these tables can be adapted in a number of ways.See Nakamura, Y., et al. “Codon usage tabulated from the internationalDNA sequence databases: status for the year 2000” Nucl. Acids Res.28:292 (2000). Computer algorithms for codon optimizing a particularsequence for expression in a particular host cell are also available,such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In someembodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50,or more, or all codons) in a sequence encoding a CRISPR enzymecorrespond to the most frequently used codon for a particular aminoacid.

In some embodiments, a vector encodes a CRISPR enzyme comprising one ormore nuclear localization sequences (NLSs), such as about or more thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments,the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near thecarboxy-terminus, or a combination of these (e.g. one or more NLS at theamino-terminus and one or more NLS at the carboxy terminus). When morethan one NLS is present, each may be selected independently of theothers, such that a single NLS may be present in more than one copyand/or in combination with one or more other NLSs present in one or morecopies. In a preferred embodiment of the invention, the CRISPR enzymecomprises at most 6 NLSs. In some embodiments, an NLS is considered nearthe N- or C-terminus when the nearest amino acid of the NLS is withinabout 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acidsalong the polypeptide chain from the N- or C-terminus. Typically, an NLSconsists of one or more short sequences of positively charged lysines orarginines exposed on the protein surface, but other types of NLS areknown. Non-limiting examples of NLSs include an NLS sequence derivedfrom: the NLS of the SV40 virus large T-antigen, having the amino acidsequence PKKKRKV (SEQ ID NO: 1); the NLS from nucleoplasmin (e.g. thenucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ IDNO: 2)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ IDNO: 3) or RQRRNELKRSP (SEQ ID NO: 4); the hRNPAI M9 NLS having thesequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) ofthe IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:7) and PPKKARED (SEQ ID NO: 8) of the myoma T protein; the sequencePQPKKKPL (SEQ ID NO: 9) of human p53; the sequence SALIKKKKKMAP (SEQ IDNO: 10) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 11) andPKQKKRK (SEQ ID NO: 12) of the influenza virus NS1; the sequenceRKLKKKIKKL (SEQ ID NO: 13) of the Hepatitis virus delta antigen; thesequence REKKKFLKRR (SEQ ID NO: 14) of the mouse Mx protein; thesequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 16) of the steroid hormone receptors (human) glucocorticoid.

In general, the one or more NLSs are of sufficient strength to driveaccumulation of the CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In general, strength of nuclear localizationactivity may derive from the number of NLSs in the CRISPR enzyme, theparticular NLS(s) used, or a combination of these factors. Detection ofaccumulation in the nucleus may be performed by any suitable technique.For example, a detectable marker may be fused to the CRISPR enzyme, suchthat location within a cell may be visualized, such as in combinationwith a means for detecting the location of the nucleus (e.g. a stainspecific for the nucleus such as DAPI). Examples of detectable markersinclude fluorescent proteins (such as Green fluorescent proteins, orGFP; RFP; CFP), and epitope tags (HA tag, flag tag, SNAP tag). Cellnuclei may also be isolated from cells, the contents of which may thenbe analyzed by any suitable process for detecting protein, such asimmunohistochemistry, Western blot, or enzyme activity assay.Accumulation in the nucleus may also be determined indirectly, such asby an assay for the effect of CRISPR complex formation (e.g. assay forDNA cleavage or mutation at the target sequence, or assay for alteredgene expression activity affected by CRISPR complex formation and/orCRISPR enzyme activity), as compared to a control no exposed to theCRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the oneor more NLSs.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). In some embodiments, a guide sequence is about ormore than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotidesin length. In some embodiments, a guide sequence is less than about 75,50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Theability of a guide sequence to direct sequence-specific binding of aCRISPR complex to a target sequence may be assessed by any suitableassay. For example, the components of a CRISPR system sufficient to forma CRISPR complex, including the guide sequence to be tested, may beprovided to a host cell having the corresponding target sequence, suchas by transfection with vectors encoding the components of the CRISPRsequence, followed by an assessment of preferential cleavage within thetarget sequence, such as by Surveyor assay as described herein.Similarly, cleavage of a target polynucleotide sequence may be evaluatedin a test tube by providing the target sequence, components of a CRISPRcomplex, including the guide sequence to be tested and a control guidesequence different from the test guide sequence, and comparing bindingor rate of cleavage at the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome. For example, for the S. pyogenes Cas9, a unique targetsequence in a genome may include a Cas9target site of the formMMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 530) where NNNNNNNNNNNNXGG (SEQ IDNO: 531) (N is A, G, T, or C; and X can be anything) has a singleoccurrence in the genome. A unique target sequence in a genome mayinclude an S. pyogenes Cas9 target site of the formMMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 532) where NNNNNNNNNNNXGG (SEQ IDNO: 533) (N is A, G, T, or C; and X can be anything) has a singleoccurrence in the genome. For the S. thermophilus CRISPR1 Cas9, a uniquetarget sequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 17) where NNNNNNNNNNNNXXAGAAW(SEQ ID NO: 18) (N is A, G, T, or C; X can be anything; and W is A or T)has a single occurrence in the genome. A unique target sequence in agenome may include an S. thermophilus CRISPR1 Cas9 target site of theform MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 19) whereNNNNNNNNNNNXXAGAAW (SEQ ID NO: 20) (N is A, G, T, or C; X can beanything; and W is A or T) has a single occurrence in the genome. Forthe S. pyogenes Cas9, a unique target sequence in a genome may include aCas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG (SEQ ID NO: 534)where NNNNNNNNNNNNXGGXG (SEQ ID NO: 535) (N is A, G, T, or C; and X canbe anything) has a single occurrence in the genome. A unique targetsequence in a genome may include an S. pyogenes Cas9 target site of theform MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 536) where NNNNNNNNNNNXGGXG(SEQ ID NO: 537) (N is A, G, T, or C; and X can be anything) has asingle occurrence in the genome. In each of these sequences “M” may beA, G, T, or C, and need not be considered in identifying a sequence asunique.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSer. No. 61/836,080 filed Jun. 17, 2013 (attorney docket 44790.11.2022;Broad Reference BI-2013/004A); incorporated herein by reference.

In general, a tracr mate sequence includes any sequence that hassufficient complementarity with a tracr sequence to promote one or moreof: (1) excision of a guide sequence flanked by tracr mate sequences ina cell containing the corresponding tracr sequence; and (2) formation ofa CRISPR complex at a target sequence, wherein the CRISPR complexcomprises the tracr mate sequence hybridized to the tracr sequence. Ingeneral, degree of complementarity is with reference to the optimalalignment of the tracr mate sequence and tracr sequence, along thelength of the shorter of the two sequences. Optimal alignment may bedetermined by any suitable alignment algorithm, and may further accountfor secondary structures, such as self-complementarity within either thetracr sequence or tracr mate sequence. In some embodiments, the degreeof complementarity between the tracr sequence and tracr mate sequencealong the length of the shorter of the two when optimally aligned isabout or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,97.5%, 99%, or higher. Example illustrations of optimal alignmentbetween a tracr sequence and a tracr mate sequence are provided in FIGS.12B and 13B. In some embodiments, the tracr sequence is about or morethan about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 40, 50, or more nucleotides in length. In some embodiments, thetracr sequence and tracr mate sequence are contained within a singletranscript, such that hybridization between the two produces atranscript having a secondary structure, such as a hairpin. Preferredloop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG. In anembodiment of the invention, the transcript or transcribedpolynucleotide sequence has at least two or more hairpins. In preferredembodiments, the transcript has two, three, four or five hairpins. In afurther embodiment of the invention, the transcript has at most fivehairpins. In some embodiments, the single transcript further includes atranscription termination sequence; preferably this is a polyT sequence,for example six T nucleotides. An example illustration of such a hairpinstructure is provided in the lower portion of FIG. 13B, where theportion of the sequence 5′ of the final “N” and upstream of the loopcorresponds to the tracr mate sequence, and the portion of the sequence3′ of the loop corresponds to the tracr sequence. Further non-limitingexamples of single polynucleotides comprising a guide sequence, a tracrmate sequence, and a tracr sequence are as follows (listed 5′ to 3′),where “N” represents a base of a guide sequence, the first block oflower case letters represent the tracr mate sequence, and the secondblock of lower case letters represent the tracr sequence, and the finalpoly-T sequence represents the transcription terminator: (1)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO:21); (2)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 22); (3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 23); (4)NNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 24); (5)NNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTT (SEQ ID NO: 25); and (6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTT (SEQ ID NO: 26). In some embodiments, sequences (1) to (3) are usedin combination with Cas9 from S. thermophilus CRISPR1. In someembodiments, sequences (4) to (6) are used in combination with Cas9 fromS. pyogenes. In some embodiments, the tracr sequence is a separatetranscript from a transcript comprising the tracr mate sequence (such asillustrated in the top portion of FIG. 13B).

In some embodiments, a recombination template is also provided. Arecombination template may be a component of another vector as describedherein, contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by a CRISPR enzyme asa part of a CRISPR complex. A template polynucleotide may be of anysuitable length, such as about or more than about 10, 15, 20, 25, 50,75, 100, 150, 200, 500, 1000, or more nucleotides in length. In someembodiments, the template polynucleotide is complementary to a portionof a polynucleotide comprising the target sequence. When optimallyaligned, a template polynucleotide might overlap with one or morenucleotides of a target sequences (e.g. about or more than about 1, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or morenucleotides). In some embodiments, when a template sequence and apolynucleotide comprising a target sequence are optimally aligned, thenearest nucleotide of the template polynucleotide is within about 1, 5,10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, ormore nucleotides from the target sequence.

In some embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Non-limiting examples ofepitope tags include histidine (His) tags, V5 tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags. VSV-G tags, and thioredoxin (Trx)tags. Examples of reporter genes include, but are not limited to,glutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP). ACRISPR enzyme may be fused to a gene sequence encoding a protein or afragment of a protein that bind DNA molecules or bind other cellularmolecules, including but not limited to maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domainfusions, and herpes simplex virus (HSV) BP16 protein fusions. Additionaldomains that may form part of a fusion protein comprising a CRISPRenzyme are described in US20110059502, incorporated herein by reference.In some embodiments, a tagged CRISPR enzyme is used to identify thelocation of a target sequence.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a CRISPR enzyme in combination with (and optionallycomplexed with) a guide sequence is delivered to a cell. Conventionalviral and non-viral based gene transfer methods can be used to introducenucleic acids in mammalian cells or target tissues. Such methods can beused to administer nucleic acids encoding components of a CRISPR systemto cells in culture, or in a host organism. Non-viral vector deliverysystems include DNA plasmids, RNA (e.g. a transcript of a vectordescribed herein), naked nucleic acid, and nucleic acid complexed with adelivery vehicle, such as a liposome. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner,TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology, Doerfler and Böhm (eds) (1995); and Yuet al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SW), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and yr2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr−/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a CRISPR system as described herein (such as by transienttransfection of one or more vectors, or transfection with RNA), andmodified through the activity of a CRISPR complex, is used to establisha new cell line comprising cells containing the modification but lackingany other exogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors described herein,or cell lines derived from such cells are used in assessing one or moretest compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. In certain embodiments, the organism or subject is a plant. Incertain embodiments, the organism or subject or plant is algae. Methodsfor producing transgenic plants and animals are known in the art, andgenerally begin with a method of cell transfection, such as describedherein.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence.

With recent advances in crop genomics, the ability to use CRISPR-Cassystems to perform efficient and cost effective gene editing andmanipulation will allow the rapid selection and comparison of single andmultiplexed genetic manipulations to transform such genomes for improvedproduction and enhanced traits. In this regard reference is made to USpatents and publications: U.S. Pat. No. 6,603,061—Agrobacterium-MediatedPlant Transformation Method; U.S. Pat. No. 7,868,149—Plant GenomeSequences and Uses Thereof and US 2009/0100536—Transgenic Plants withEnhanced Agronomic Traits, all the contents and disclosure of each ofwhich are herein incorporated by reference in their entirety. In thepractice of the invention, the contents and disclosure of Morrell et al“Crop genomics:advances and applications” Nat Rev Genet. 2011 Dec. 29;13(2):85-96 are also herein incorporated by reference in their entirety.In an advantageous embodiment of the invention, the CRISPR/Cas9 systemis used to engineer microalgae (Example 15). Accordingly, referenceherein to animal cells may also apply, mutatis mutandis, to plant cellsunless otherwise apparent.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal or plant (includingmicro-algae), and modifying the cell or cells. Culturing may occur atany stage ex vivo. The cell or cells may even be re-introduced into thenon-human animal or plant (including micro-algae).

In plants, pathogens are often host-specific. For example, Fusariumnoxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f. dianthii Puccinia graminis f. sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield.Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises a vector system and instructions forusing the kit. In some embodiments, the vector system comprises (a) afirst regulatory element operably linked to a tracr mate sequence andone or more insertion sites for inserting a guide sequence upstream ofthe tracr mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aCRISPR enzyme complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the tracr mate sequence that ishybridized to the tracr sequence; and/or (b) a second regulatory elementoperably linked to an enzyme-coding sequence encoding said CRISPR enzymecomprising a nuclear localization sequence. Elements may be provideindividually or in combinations, and may be provided in any suitablecontainer, such as a vial, a bottle, or a tube. In some embodiments, thekit includes instructions in one or more languages, for example in morethan one language.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utility includingmodifying (e.g., deleting, inserting, translocating, inactivating,activating) a target polynucleotide in a multiplicity of cell types. Assuch the CRISPR complex of the invention has a broad spectrum ofapplications in, e.g., gene therapy, drug screening, disease diagnosis,and prognosis. An exemplary CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinthe target polynucleotide. The guide sequence is linked to a tracr matesequence, which in turn hybridizes to a tracr sequence.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif): that is, a short sequencerecognized by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are given inthe examples section below, and the skilled person will be able toidentify further PAM sequences for use with a given CRISPR enzyme.

The target polynucleotide of a CRISPR complex may include a number ofdisease-associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes and polynucleotides as listed in USprovisional patent applications 61/736,527 and 61/748,427 having Broadreference B1-2011/008/WSGR Docket No. 44063-701.101 and B1-2011/008/WSGRDocket No. 44063-701.102 respectively, both entitled SYSTEMS METHODS ANDCOMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan.2, 2013, respectively, the contents of all of which are hereinincorporated by reference in their entirety.

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

Examples of disease-associated genes and polynucleotides are availablefrom McKusick-Nathans Institute of Genetic Medicine, Johns HopkinsUniversity (Baltimore, Md.) and National Center for BiotechnologyInformation, National Library of Medicine (Bethesda, Md.), available onthe World Wide Web.

Examples of disease-associated genes and polynucleotides are listed inTables A and B. Disease specific information is available fromMcKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University(Baltimore. Md.) and National Center for Biotechnology Information,National Library of Medicine (Bethesda, Md.), available on the WorldWide Web. Examples of signaling biochemical pathway-associated genes andpolynucleotides are listed in Table C.

Mutations in these genes and pathways can result in production ofimproper proteins or proteins in improper amounts which affect function.Further examples of genes, diseases and proteins are hereby incorporatedby reference from U.S. Provisional application 61/736,527 filed on Dec.12, 2012 and 61/748,427 filed on Feb. 2, 2013. Such genes, proteins andpathways may be the target polynucleotide of a CRISPR complex.

TABLE A DISEASE/DISORDERS GENE(S) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2;ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF;HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor);FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB(retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (Androgen Receptor);TSG101; IGF; IGF Receptor; Igf1 (4 variants); Igf2 (3 variants); Igf 1Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2,3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Macular Abcr; Ccl2; Cc2; cp(ceruloplasmin); Timp3; cathepsinD; Degeneration Vldlr; Ccr2Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin);Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophanhydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT (Slc6a4);COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1) Trinucleotide RepeatHTT (Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Disorders Dx); FXN/X25(Friedrich's Ataxia); ATX3 (Machado- Joseph's Dx); ATXN1 and ATXN2(spinocerebellar ataxias); DMPK (myotonic dystrophy); Atrophin-1 andAtn1 (DRPLA Dx); CBP (Creb-BP - global instability); VLDLR(Alzheimer's); Atxn7; Atxn10 Fragile X Syndrome FMR2; FXR1; FXR2; mGLUR5Secretase Related APH-1 (alpha and beta); Presenilin (Psen1); nicastrinDisorders (Ncstn); PEN-2 Others Nos1; Parp1; Nat1; Nat2 Prion - relateddisorders Prp ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b;VEGF-c) Drug addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol);GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) AutismMecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1;FXR2; Mglur5) Alzheimer's Disease E1; CHIP; UCH; UBB; Tau; LRP; PICALM;Clusterin; PS1; SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin1); Uchl1; Uchl3; APP Inflammation IL-10; IL-1 (IL-1a; IL-1b); IL-13;IL-17 (IL-17a (CTLA8); IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1;ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4;Cx3cl1 Parkinson's Disease x-Synuclein; DJ-1; LRRK2; Parkin; PINK1

TABLE B Blood and Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3,UMPH1, coagulation diseases PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2,ANH1, ASB, and disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome(TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP,RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H and factorH-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VIIdeficiency (F7); Factor X deficiency (F10); Factor XI deficiency (F11);Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A);Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA,FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1,FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocyticlymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3,HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB),Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies anddisorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3,EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia(HBA2, HBB, HBD, LCRB, HBA1). Cell dysregulation B-cell non-Hodgkinlymphoma (BCL7A, BCL7); Leukemia (TAL1, and oncology TCL5, SCL, TAL2,FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases and disorders HOXD4,HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12,LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT,LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3,FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM,CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF,WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA,GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN,CAIN). Inflammation and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1,IFNG, CXCL12, immune related SDF1); Autoimmune lymphoproliferativesyndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A);Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1 (CCL5,SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF,CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G,AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG,HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI);Inflammation (IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8),IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa,NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1);Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS,SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG,SCIDX1, SCIDX, IMD4). Metabolic, liver, Amyloid neuropathy (TTR, PALB);Amyloidosis (APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN, FGA,LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, diseases and disorders CIRH1A,NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7);Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2,LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1,HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder(SCOD1, SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancerand carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53,P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidneydisease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1,QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1,ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).Muscular/Skeletal Becker muscular dystrophy (DMD, BMD, MYF6), DuchenneMuscular diseases and disorders Dystrophy (DMD, BMD); Emery-Dreifussmuscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA,LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy(FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM,LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B,SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E,SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H,FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C,SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1,LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7,OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2,SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2,CATF1, SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP,VEGF (VEGF-a, VEGF-b, neuronal diseases and VEGF-c); Alzheimer disease(APP, AAA, CVAP, AD1, APOE, AD2, disorders PSEN2, AD4, STM2, APBB2,FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP,A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A,Neurexin1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260,AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5); Huntington'sdisease and disease like disorders (HD, IT15, PRNP, PRIP, JPH3, JP3,HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT, TINUR, SNCAIP,TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2, PARK8, PINK1,PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH,NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9,MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1); Schizophrenia(Neuregulin1 (Nrg1), Erb4 (receptor for Neuregulin), Complexin1 (Cplx1),Tph1 Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1,GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA,DTNBP1, Dao (Dao1)); Secretase Related Disorders (APH-1 (alpha andbeta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Nat1,Nat2); Trinucleotide Repeat Disorders (HTT (Huntington's Dx),SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3(Machado-Joseph's Dx), ATXN1 and ATXN2 (spinocerebellar ataxias), DMPK(myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP(Creb-BP-global instability), VLDLR (Alzheimer's), Atxn7, Atxn10).Occular diseases and Age-related macular degeneration (Abcr, Ccl2, Cc2,cp (ceruloplasmin), disorders Timp3, cathepsinD, Vldlr, Ccr2); Cataract(CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1,PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD,CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2,CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA,CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1);Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3,CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD,PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma(MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1,GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1,RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4,GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4,ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

TABLE C CELLULAR FUNCTION GENES PI3K/AKT Signaling PRKCE; ITGAM; ITGA5;IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1;AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8;BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1;MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB;DYRK1A; CDKN1A; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1;PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN;ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2;RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA;CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8;MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9;SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1;FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3;ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF;STAT1; SGK Glucocorticoid Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6;PCAF; ELK1; Signaling MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA;CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8;BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A;MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3;MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8;NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1;SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 AxonalGuidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; Signaling IGF1;RAC1; RAP1A; EIF4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF;RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ;PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS;RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2;PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3;CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA EphrinReceptor PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2;EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1;AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8;GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2;PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; AKT1; JAK2;STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK;CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK Actin Cytoskeleton ACTN4;PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; INS;ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1;PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS;RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN;VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1;PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGKHuntington's Disease PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2;Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5;CREB1; PRKCI; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1;GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11;MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1;CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK;HDAC6; CASP3 Apoptosis Signaling PRKCE; ROCK1; BID; IRAK1; PRKAA2;EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2;CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8;KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG;RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA;CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 BCell Receptor RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; SignalingAKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3;MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9;EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1;PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN;GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte Extravasation ACTN4; CD44;PRKCE; ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A; PRKCZ; ROCK2;RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8;PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A;BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1;CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9Integrin Signaling ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1;ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3;MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7;PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1;TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11;Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8;RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1;TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2;AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3;IL1R1; IL6 PTEN Signaling ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11;MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2;PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1;IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1;MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1;CASP3; RPS6KB1 p53 Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1;GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3;MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1;HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1;RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2;GSK3B; BAX; AKT3 Aryl Hydrocarbon HSPB1; EP300; FASN; TGM2; RXRA; MAPK1;NQO1; Receptor Signaling NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; SMARCA4;NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73;GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2;APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6;CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE; EP300; PRKCZ; RXRA; MAPK1;NQO1; Signaling NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB;PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13;PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A;PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1;NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK SignalingPRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2;PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1;IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1;PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3;CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling PRKAA2;EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB;NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS;RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7;CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1;PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB Signaling IRAK1; EIF2AK2; EP300; INS;MYD88; PRKCZ; TRAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2;MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A;TRAF2; TLR4; PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1;PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1Neuregulin Signaling ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1;MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3;ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17;AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC;NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44;EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC;GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2; ILK; LEF1;SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1;TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2Insulin Receptor PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; SignalingPTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3;TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2;JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B;AKT3; FOXO1; SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1;MAPK1; PTPN11; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST;KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1;IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1;CEBPB; JUN; IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS;MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8;PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG;RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN;IL1R1; PRKCA; IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11;NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R;IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2;AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF;CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1;Oxidative Stress Response NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A;MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN;KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic Fibrosis/HepaticEDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; Stellate Cell ActivationSMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4;PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1;CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS;TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B;MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF;INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1;NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon RI Signaling PRKCE; RAC1; PRKCZ;LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK;MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3;PRKCA G-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB;Receptor Signaling PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3;MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1;PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCAInositol Phosphate PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MetabolismMAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD;PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1;MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1;ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3;KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA;STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGFSignaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA;ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3;PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA;AKT3; FOXO1; PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2;PTPN11; Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4;AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4;SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation ATR; ABL1;E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53;CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1;HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS;Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA; PIK3C2A; BTK;LCK; RAF1; IKBKG; RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK;BCL10; JUN; VAV3 Death Receptor Signaling CRADD; HSPB1; BID; BIRC4;TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX;TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1;CASP2; BIRC2; CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2;MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3;MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1;FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GM-CSF Signaling LYN; ELK1; MAPK1;PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1;MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID; IGF1;RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA; BCL2;PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A;CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat SignalingPTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS;SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1;PRKAA2; EIF2AK2; GRK6; MAPK1; Nicotinamide Metabolism PLK1; AKT2; CDK8;MAPK8; MAPK3; PRKCD; PRKAA1; PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2;MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine Signaling CXCR4;ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS;MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1;JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK;FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A;LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 Synaptic LongTerm PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1; GNAS; Depression PRKCI;GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A;PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA Estrogen ReceptorTAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; Signaling SMARCA4; MAPK3;NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP;MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 Protein Ubiquitination TRAF6;SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; Pathway CBL; UBE2I; BTRC; HSPA5;USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1;VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1; ELK1; IKBKB; SP1; FOS;NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7;JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXR Activation PRKCE;EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1;PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1;PRKCA TGF-beta Signaling EP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; FOS;MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1; RAF1; MAP3K7; CREBBP;MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Toll-like Receptor IRAK1;EIF2AK2; MYD88; TRAF6; PPARA; ELK1; Signaling IKBKB; FOS; NFKB2;MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK;NFKB1; TLR2; JUN p38 MAPK Signaling HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1;FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF;MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1 Neurotrophin/TRK NTRK2;MAPK1; PTPN11; PIK3CA; CREB1; FOS; Signaling PIK3CB; PIK3C3; MAPK8;MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42;JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8;APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1;FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE; RAP1A; EP300; PRKCZ; MAPK1;CREB1; Potentiation PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; PRKCD;PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4; PRKCA Calcium SignalingRAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2;HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; HDAC6 EGFSignaling ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3;PIK3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1Hypoxia Signaling in the EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT;Cardiovascular System HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; VEGFA;JUN; ATF4; VHL; HSP90AA1 LPS/IL-1 Mediated IRAK1; MYD88; TRAF6; PPARA;RXRA; ABCA1; Inhibition of RXR Function MAPK8; ALDH1A1; GSTP1; MAPK9;ABCB1; TRAF2; TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1 LXR/RXRActivation FASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA; NOS2A; TLR4;TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 AmyloidProcessing PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3;MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP IL-4Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1; KRAS; SOCS1; PTPN6; NR3C1;PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KB1 Cell Cycle: G2/MDNA EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; Damage Checkpoint CHEK1;ATR; CHEK2; YWHAZ; TP53; CDKN1A; Regulation PRKDC; ATM; SFN; CDKN2ANitric Oxide Signaling in KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3;the Cardiovascular System CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1;VEGFA; AKT3; HSP90AA1 Purine Metabolism NME2; SMARCA4; MYH9; RRM2; ADAR;EIF2AK4; PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1cAMP-mediated RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3; Signaling SRC;RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8;CASP8; MAPK10; MAPK9; CASP9; Dysfunction PARK7; PSEN1; PARK2; APP; CASP3Notch Signaling HES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3;NOTCH1; DLL4 Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6;CASP9; ATF4; Stress Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2;AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson'sSignaling UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3Cardiac & Beta GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; AdrenergicSignaling PPP2R5C Glycolysis/Gluconeogenesis HK2; GCK; GPI; ALDH1A1;PKM2; LDHA; HK1 Interferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1;STAT1; IFIT3 Sonic Hedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B;DYRK1B Signaling Glycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1;SPHK2 Metabolism Phospholipid PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2Degradation Tryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1;SIAH1 Lysine Degradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C NucleotideExcision ERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and SucroseUCHL1; HK2; GCK; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2;GCK; HK1 Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism CircadianRhythm CSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1;F2R; SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5CSignaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1 GlycerolipidMetabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid PRDX6; GRN; YWHAZ;CYP1B1 Metabolism Methionine Metabolism DNMT1; DNMT3B; AHCY; DNMT3APyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and ProlineALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN; YWHAZFructose and Mannose HK2; GCK; HK1 Metabolism Galactose Metabolism HK2;GCK; HK1 Stilbene, Coumarine and PRDX6; PRDX1; TYR Lignin BiosynthesisAntigen Presentation CALR; B2M Pathway Biosynthesis of Steroids NQO1;DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 FattyAcid Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKAMetabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol MetabolismERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome p450Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1 MetabolismPropanoate Metabolism ALDH1A1; LDHA Selenoamino Acid PRMT5; AHCYMetabolism Sphingolipid Metabolism SPHK1; SPHK2 Aminophosphonate PRMT5Metabolism Androgen and Estrogen PRMT5 Metabolism Ascorbate and AldarateALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1 Cysteine MetabolismLDHA Fatty Acid Biosynthesis FASN Glutamate Receptor GNB2L1 SignalingNRF2-mediated PRDX1 Oxidative Stress Response Pentose Phosphate GPIPathway Pentose and Glucuronate UCHL1 Interconversions RetinolMetabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5,TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1 IsoleucineDegradation Glycine, Serine and CHKA Threonine Metabolism LysineDegradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6;TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5;Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF; CytC; SMAC(Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Noggin(Nog); WNT (Wnt2; Neurology Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b;Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1;Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86(Pou4f1 or Brn3a); Numb; Reln

Embodiments of the invention also relate to methods and compositionsrelated to knocking out genes, amplifying genes and repairing particularmutations associated with DNA repeat instability and neurologicaldisorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities andNeurological Diseases, Second Edition, Academic Press, Oct. 13,2011—Medical). Specific aspects of tandem repeat sequences have beenfound to be responsible for more than twenty human diseases (Newinsights into repeat instability: role of RNA*DNA hybrids. McIvor E I,Polak U, Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). TheCRISPR-Cas system may be harnessed to correct these defects of genomicinstability.

A further aspect of the invention relates to utilizing the CRISPR-Cassystem for correcting defects in the EMP2A and EMP2B genes that havebeen identified to be associated with Lafora disease. Lafora disease isan autosomal recessive condition which is characterized by progressivemyoclonus epilepsy which may start as epileptic seizures in adolescence.A few cases of the disease may be caused by mutations in genes yet to beidentified. The disease causes seizures, muscle spasms, difficultywalking, dementia, and eventually death. There is currently no therapythat has proven effective against disease progression. Other geneticabnormalities associated with epilepsy may also be targeted by theCRISPR-Cas system and the underlying genetics is further described inGenetics of Epilepsy and Genetic Epilepsies, edited by GiulianoAvanzini, Jeffrey L. Noebels, Mariani Foundation PaediatricNeurology:20; 2009).

In yet another aspect of the invention, the CRISPR-Cas system may beused to correct ocular defects that arise from several genetic mutationsfurther described in Genetic Diseases of the Eye, Second Edition, editedby Elias I. Traboulsi, Oxford University Press, 2012.

Several further aspects of the invention relate to correcting defectsassociated with a wide range of genetic diseases which are furtherdescribed on the website of the National Institutes of Health under thetopic subsection Genetic Disorders (website athealth.nih.gov/topic/GeneticDisorders). The genetic brain diseases mayinclude but are not limited to Adrenoleukodystrophy, Agenesis of theCorpus Callosum, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease,Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration,Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington'sDisease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-NyhanSyndrome, Menkes Disease, Mitochondrial Myopathies and NINDSColpocephaly. These diseases are further described on the website of theNational Institutes of Health under the subsection Genetic BrainDisorders.

In some embodiments, the condition may be neoplasia. In someembodiments, where the condition is neoplasia, the genes to be targetedare any of those listed in Table A (in this case PTEN and so forth). Insome embodiments, the condition may be Age-related Macular Degeneration.In some embodiments, the condition may be a Schizophrenic Disorder. Insome embodiments, the condition may be a Trinucleotide Repeat Disorder.In some embodiments, the condition may be Fragile X Syndrome. In someembodiments, the condition may be a Secretase Related Disorder. In someembodiments, the condition may be a Prion-related disorder. In someembodiments, the condition may be ALS. In some embodiments, thecondition may be a drug addiction. In some embodiments, the conditionmay be Autism. In some embodiments, the condition may be Alzheimer'sDisease. In some embodiments, the condition may be inflammation. In someembodiments, the condition may be Parkinson's Disease.

Examples of proteins associated with Parkinson's disease include but arenot limited to α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1,Synphilin-1, and NURR1.

Examples of addiction-related proteins may include ABAT for example.

Examples of inflammation-related proteins may include the monocytechemoattractant protein-1 (MCP1) encoded by the Ccr2 gene, the C-Cchemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, the IgGreceptor IIB (FCGR2b, also termed CD32) encoded by the Fcgr2b gene, orthe Fc epsilon R1g (FCER1g) protein encoded by the Fcer1g gene, forexample.

Examples of cardiovascular diseases associated proteins may include IL1B(interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor proteinp53). PTGIS (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin),IL4 (interleukin 4), ANGPTI (angiopoietin 1). ABCG8 (ATP-bindingcassette, sub-family G (WHITE), member 8), or CTSK (cathepsin K), forexample.

Examples of Alzheimer's disease associated proteins may include the verylow density lipoprotein receptor protein (VLDLR) encoded by the VLDLRgene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded bythe UBA1 gene, or the NEDD8-activating enzyme E1 catalytic subunitprotein (UBEIC) encoded by the UBA3 gene, for example.

Examples of proteins associated Autism Spectrum Disorder may include thebenzodiazapine receptor (peripheral) associated protein 1 (BZRAPI)encoded by the BZRAPI gene, the AF4/FMR2 family member 2 protein (AFF2)encoded by the AFF2 gene (also termed MFR2), the fragile X mentalretardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene,or the fragile X mental retardation autosomal homolog 2 protein (FXR2)encoded by the FXR2 gene, for example.

Examples of proteins associated Macular Degeneration may include theATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4)encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded bythe APOE gene, or the chemokine (C-C motif) Ligand 2 protein (CCL2)encoded by the CCL2 gene, for example.

Examples of proteins associated Schizophrenia may include NRG1, ErbB4,CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC 1, GSK3B, and combinationsthereof.

Examples of proteins involved in tumor suppression may include ATM(ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-crb-b2erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch2,Notch 3, or Notch 4, for example.

Examples of proteins associated with a secretase disorder may includePSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B),PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH 1B(anterior pharynx defective 1 homolog B (C. elegans)), PSEN2 (presenilin2 (Alzheimer disease 4)), or BACE1 (beta-site APP-cleaving enzyme 1),for example.

Examples of proteins associated with Amyotrophic Lateral Sclerosis mayinclude SOD1 (superoxide dismutase 1). A LS2 (amyotrophic lateralsclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein),VAGFA (vascular endothelial growth factor A), VAGFB (vascularendothelial growth factor B), and VAGFC (vascular endothelial growthfactor C), and any combination thereof.

Examples of proteins associated with prion diseases may include SOD1(superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS(fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascularendothelial growth factor A), VAGFB (vascular endothelial growth factorB), and VAGFC (vascular endothelial growth factor C), and anycombination thereof.

Examples of proteins related to neurodegenerative conditions in priondisorders may include A2M (Alpha-2-Macroglobulin), AATF (Apoptosisantagonizing transcription factor), ACPP (Acid phosphatase prostate).ACTA2 (Actin alpha 2 smooth muscle aorta), ADAM22 (ADAM metallopeptidasedomain), ADORA3 (Adenosine A3 receptor), or ADRA1D (Alpha-1D adrenergicreceptor for Alpha-1D adrenoreceptor), for example.

Examples of proteins associated with Immunodeficiency may include A2M[alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase];ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1]; ABCA2[ATP-binding cassette, sub-family A (ABC1), member 2]; or ABCA3[ATP-binding cassette, sub-family A (ABC1), member 3]; for example.

Examples of proteins associated with Trinucleotide Repeat Disordersinclude AR (androgen receptor), FMR1 (fragile X mental retardation 1),HTT (huntingtin), or DMPK (dystrophia myotonica-protein kinase), FXN(frataxin), ATXN2 (ataxin 2), for example.

Examples of proteins associated with Neurotransmission Disorders includeSST (somatostatin). NOS1 (nitric oxide synthase 1 (neuronal)), ADRA2A(adrenergic, alpha-2A-, receptor), ADRA2C (adrenergic, alpha-2C-,receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine(serotonin) receptor 2C), for example.

Examples of neurodevelopmental-associated sequences include A2BP1[ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase],AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyrateaminotransferase], ABCA1 [ATP-binding cassette, sub-family A (ABC1),member 1], or ABCA13 [ATP-binding cassette, sub-family A (ABC1), member13], for example.

Further examples of preferred conditions treatable with the presentsystem include may be selected from: Aicardi-Goutières Syndrome;Alexander Disease; Allan-Herndon-Dudley Syndrome; POLG-RelatedDisorders; Alpha-Mannosidosis (Type II and III); Alström Syndrome;Angelman; Syndrome; Ataxia-Telangiectasia; NeuronalCeroid-Lipofuscinoses; Beta-Thalassemia; Bilateral Optic Atrophy and(Infantile) Optic Atrophy Type 1; Retinoblastoma (bilateral); CanavanDisease; Cerebrooculofacioskeletal Syndrome 1 [COFS1]; CerebrotendinousXanthomatosis; Cornelia de Lange Syndrome; MAPT-Related Disorders;Genetic Prion Diseases; Dravet Syndrome; Early-Onset Familial AlzheimerDisease; Friedreich Ataxia [FRDA]; Fryns Syndrome; Fucosidosis; FukuyamaCongenital Muscular Dystrophy; Galactosialidosis; Gaucher Disease;Organic Acidemias; Hemophagocytic Lymphohistiocytosis;Hutchinson-Gilford Progeria Syndrome; Mucolipidosis II; Infantile FreeSialic Acid Storage Disease; PLA2G6-Associated Neurodegeneration;Jervell and Lange-Nielsen Syndrome; Junctional Epidermolysis Bullosa;Huntington Disease; Krabbe Disease (Infantile); MitochondrialDNA-Associated Leigh Syndrome and NARP; Lesch-Nyhan Syndrome;LIS1-Associated Lissencephaly; Lowe Syndrome; Maple Syrup Urine Disease;MECP2 Duplication Syndrome; ATP7A-Related Copper Transport Disorders;LAMA2-Related Muscular Dystrophy; Arylsulfatase A Deficiency;Mucopolysaccharidosis Types I, II or III; Peroxisome BiogenesisDisorders, Zellweger Syndrome Spectrum; Neurodegeneration with BrainIron Accumulation Disorders; Acid Sphingomyelinase Deficiency;Niemann-Pick Disease Type C; Glycine Encephalopathy; ARX-RelatedDisorders; Urea Cycle Disorders; COL1A1/2-Related OsteogenesisImperfecta; Mitochondrial DNA Deletion Syndromes; PLP1-RelatedDisorders; Perry Syndrome; Phelan-McDermid Syndrome; Glycogen StorageDisease Type II (Pompe Disease) (Infantile); MAPT-Related Disorders;MECP2-Related Disorders; Rhizomelic Chondrodysplasia Punctata Type 1;Roberts Syndrome; Sandhoff Disease; Schindler Disease—Type 1; AdenosineDeaminase Deficiency; Smith-Lemli-Opitz Syndrome; Spinal MuscularAtrophy; Infantile-Onset Spinocerebellar Ataxia; Hexosaminidase ADeficiency; Thanatophoric Dysplasia Type 1; Collagen Type VI-RelatedDisorders; Usher Syndrome Type I; Congenital Muscular Dystrophy;Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase Deficiency; andXeroderma Pigmentosum.

As will be apparent, it is envisaged that the present system can be usedto target any polynucleotide sequence of interest. Some examples ofconditions or diseases that might be usefully treated using the presentsystem are included in the Tables above and examples of genes currentlyassociated with those conditions are also provided there. However, thegenes exemplified are not exhaustive.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1 CRISPR Complex Activity in the Nucleus of a Eukaryotic Cell

An example type II CRISPR system is the type II CRISPR locus fromStreptococcus pyogenes SF370, which contains a cluster of four genesCas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements,tracrRNA and a characteristic array of repetitive sequences (directrepeats) interspaced by short stretches of non-repetitive sequences(spacers, about 30 bp each). In this system, targeted DNA double-strandbreak (DSB) is generated in four sequential steps (FIG. 2A). First, twonon-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed fromthe CRISPR locus. Second, tracrRNA hybridizes to the direct repeats ofpre-crRNA, which is then processed into mature crRNAs containingindividual spacer sequences. Third, the mature crRNA:tracrRNA complexdirects Cas9 to the DNA target consisting of the protospacer and thecorresponding PAM via heteroduplex formation between the spacer regionof the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage oftarget DNA upstream of PAM to create a DSB within the protospacer (FIG.2A). This example describes an example process for adapting thisRNA-programmable nuclease system to direct CRISPR complex activity inthe nuclei of eukaryotic cells.

Cell Culture and Transfection

Human embryonic kidney (HEK) cell line HEK 293FT (Life Technologies) wasmaintained in Dulbecco's modified Eagle's Medium (DMEM) supplementedwith 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (LifeTechnologies), 100U/mL penicillin, and 100 μg/mL streptomycin at 37° C.with 5% CO₂ incubation. Mouse neuro2A (N2A) cell line (ATCC) wasmaintained with DMEM supplemented with 5% fetal bovine serum (HyClone),2 mM GlutaMAX (Life Technologies), 100U/mL penicillin, and 100 μg/mLstreptomycin at 37° C. with 5% CO₂.

HEK 293FT or N2A cells were seeded into 24-well plates (Corning) one dayprior to transfection at a density of 200,000 cells per well. Cells weretransfected using Lipofectamine 2000 (Life Technologies) following themanufacturer's recommended protocol. For each well of a 24-well plate atotal of 800 ng of plasmids were used.

Surveyor Assay and Sequencing Analysis for Genome Modification

HEK 293FT or N2A cells were transfected with plasmid DNA as describedabove. After transfection, the cells were incubated at 37° C. for 72hours before genomic DNA extraction. Genomic DNA was extracted using theQuickExtract DNA extraction kit (Epicentre) following the manufacturer'sprotocol. Briefly, cells were resuspended in QuickExtract solution andincubated at 65° C. for 15 minutes and 98° C. for 10 minutes. Extractedgenomic DNA was immediately processed or stored at −20° C.

The genomic region surrounding a CRISPR target site for each gene wasPCR amplified, and products were purified using QiaQuick Spin Column(Qiagen) following manufacturer's protocol. A total of 400 ng of thepurified PCR products were mixed with 2 μl 10× Taq polymerase PCR buffer(Enzymatics) and ultrapure water to a final volume of 20 μl, andsubjected to a re-annealing process to enable heteroduplex formation:95° C. for 10 min, 95° C. to 85° C. ramping at −2° C./s, 85° C. to 25°C. at −0.25° C./s, and 25° C. hold for 1 minute. After re-annealing,products were treated with Surveyor nuclease and Surveyor enhancer S(Transgenomics) following the manufacturer's recommended protocol, andanalyzed on 4-20% Novex TBE poly-acrylamide gels (Life Technologies).Gels were stained with SYBR Gold DNA stain (Life Technologies) for 30minutes and imaged with a Gel Doc gel imaging system (Bio-rad).Quantification was based on relative band intensities, as a measure ofthe fraction of cleaved DNA. FIG. 8 provides a schematic illustration ofthis Surveyor assay.

Restriction Fragment Length Polymorphism Assay for Detection ofHomologous Recombination

HEK 293FT and N2A cells were transfected with plasmid DNA, and incubatedat 37° C. for 72 hours before genomic DNA extraction as described above.The target genomic region was PCR amplified using primers outside thehomology arms of the homologous recombination (HR) template. PCRproducts were separated on a 1% agarose gel and extracted with MinEluteGelExtraction Kit (Qiagen). Purified products were digested with HindIII(Fermentas) and analyzed on a 6% Novex TBE poly-acrylamide gel (LifeTechnologies).

RNA Secondary Structure Prediction and Analysis

RNA secondary structure prediction was performed using the onlinewebserver RNAfold developed at Institute for Theoretical Chemistry atthe University of Vienna, using the centroid structure predictionalgorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and G M Church, 2009, Nature Biotechnology 27(12): 1151-62).

Bacterial Plasmid Transformation Interference Assay

Elements of the S. pyogenes CRISPR locus 1 sufficient for CRISPRactivity were reconstituted in E. coli using pCRISPR plasmid(schematically illustrated in FIG. 10A). pCRISPR contained tracrRNA,SpCas9, and a leader sequence driving the crRNA array. Spacers (alsoreferred to as “guide sequences”) were inserted into the crRNA arraybetween BsaI sites using annealed oligonucleotides, as illustrated.Challenge plasmids used in the interference assay were constructed byinserting the protospacer (also referred to as a “target sequence”)sequence along with an adjacent CRISPR motif sequence (PAM) into pUC19(see FIG. 10B). The challenge plasmid contained ampicillin resistance.FIG. 10C provides a schematic representation of the interference assay.Chemically competent E. coli strains already carrying pCRISPR and theappropriate spacer were transformed with the challenge plasmidcontaining the corresponding protospacer-PAM sequence. pUC19 was used toassess the transformation efficiency of each pCRISPR-carrying competentstrain. CRISPR activity resulted in cleavage of the pPSP plasmidcarrying the protospacer, precluding ampicillin resistance otherwiseconferred by pUC19 lacking the protospacer. FIG. 10D illustratescompetence of each pCRISPR-carrying E. coli strain used in assaysillustrated in FIG. 4C.

RNA Purification

HEK 293FT cells were maintained and transfected as stated above. Cellswere harvested by trypsinization followed by washing in phosphatebuffered saline (PBS). Total cell RNA was extracted with TRI reagent(Sigma) following manufacturer's protocol. Extracted total RNA wasquantified using Naonodrop (Thermo Scientific) and normalized to sameconcentration.

Northern Blot Analysis of crRNA and tracrRNA Expression in MammalianCells

RNAs were mixed with equal volumes of 2× loading buffer (Ambion), heatedto 95° C. for 5 min, chilled on ice for 1 min, and then loaded onto 8%denaturing polyacrylamide gels (SequaGel, National Diagnostics) afterpre-running the gel for at least 30 minutes. The samples wereelectrophoresed for 1.5 hours at 40W limit. Afterwards, the RNA wastransferred to Hybond N+ membrane (GE Healthcare) at 300 mA in asemi-dry transfer apparatus (Bio-rad) at room temperature for 1.5 hours.The RNA was crosslinked to the membrane using autocrosslink button onStratagene UV Crosslinker the Stratalinker (Stratagene). The membranewas pre-hybridized in ULTRAhyb-Oligo Hybridization Buffer (Ambion) for30 min with rotation at 42° C., and probes were then added andhybridized overnight. Probes were ordered from IDT and labeled with[gamma-³²P]ATP (Perkin Elmer) with T4 polynucleotide kinase (New EnglandBiolabs). The membrane was washed once with pre-warmed (42° C.) 2×SSC,0.5% SDS for 1 min followed by two 30 minute washes at 42° C. Themembrane was exposed to a phosphor screen for one hour or overnight atroom temperature and then scanned with a phosphorimager (Typhoon).

Bacterial CRISPR System Construction and Evaluation

CRISPR locus elements, including tracrRNA, Cas9, and leader were PCRamplified from Streptococcus pyogenes SF370 genomic DNA with flankinghomology arms for Gibson Assembly. Two BsaI type IIS sites wereintroduced in between two direct repeats to facilitate easy insertion ofspacers (FIG. 9). PCR products were cloned into EcoRV-digested pACYC184downstream of the tet promoter using Gibson Assembly Master Mix (NEB).Other endogenous CRISPR system elements were omitted, with the exceptionof the last 50 bp of Csn2. Oligos (Integrated DNA Technology) encodingspacers with complimentary overhangs were cloned into the BsaI-digestedvector pDC000 (NEB) and then ligated with T7 ligase (Enzymatics) togenerate pCRISPR plasmids. Challenge plasmids containing spacers withPAM sequences (also referred to herein as “CRISPR motif sequences”) werecreated by ligating hybridized oligos carrying compatible overhangs(Integrated DNA Technology) into BamHI-digested pUC19. Cloning for allconstructs was performed in E. coli strain JM109 (Zymo Research).

pCRISPR-carrying cells were made competent using the Z-Competent E. coliTransformation Kit and Buffer Set (Zymo Research, T3001) according tomanufacturer's instructions. In the transformation assay, 50 uL aliquotsof competent cells carrying pCRISPR were thawed on ice and transformedwith 1 ng of spacer plasmid or pUC19 on ice for 30 minutes, followed by45 second heat shock at 42° C. and 2 minutes on ice. Subsequently. 250ul SOC (Invitrogen) was added followed by shaking incubation at 37° C.for 1 hr, and 100 uL of the post-SOC outgrowth was plated onto doubleselection plates (12.5 ug/ml chloramphenicol, 100 ug/ml ampicillin). Toobtain cfu/ng of DNA, total colony numbers were multiplied by 3.

To improve expression of CRISPR components in mammalian cells, two genesfrom the SF370 locus 1 of Streptococcus pyogenes (S. pyogenes) werecodon-optimized, Cas9 (SpCas9) and RNase III (SpRNase III). Tofacilitate nuclear localization, a nuclear localization signal (NLS) wasincluded at the amino (N)- or carboxyl (C)-termini of both SpCas9 andSpRNase III (FIG. 2B). To facilitate visualization of proteinexpression, a fluorescent protein marker was also included at the N- orC-termini of both proteins (FIG. 2B). A version of SpCas9 with an NLSattached to both N- and C-termini (2×NLS-SpCas9) was also generated.Constructs containing NLS-fused SpCas9 and SpRNase III were transfectedinto 293FT human embryonic kidney (HEK) cells, and the relativepositioning of the NLS to SpCas9 and SpRNase III was found to affecttheir nuclear localization efficiency. Whereas the C-terminal NLS wassufficient to target SpRNase III to the nucleus, attachment of a singlecopy of these particular NLS's to either the N- or C-terminus of SpCas9was unable to achieve adequate nuclear localization in this system. Inthis example, the C-terminal NLS was that of nucleoplasmin(KRPAATKKAGQAKKKK (SEQ ID NO: 2)), and the C-terminal NLS was that ofthe SV40 large T-antigen (PKKKRKV (SEQ ID NO: 1)). Of the versions ofSpCas9 tested, only 2×NLS-SpCas9 exhibited nuclear localization (FIG.2B).

The tracrRNA from the CRISPR locus of S. pyogenes SF370 has twotranscriptional start sites, giving rise to two transcripts of89-nucleotides (nt) and 171 nt that are subsequently processed intoidentical 75 nt mature tracrRNAs. The shorter 89 nt tracrRNA wasselected for expression in mammalian cells (expression constructsillustrated in FIG. 7A, with functionality as determined by results ofthe Surveyor assay shown in FIG. 7B). Transcription start sites aremarked as +1, and transcription terminator and the sequence probed bynorthern blot are also indicated. Expression of processed tracrRNA wasalso confirmed by Northern blot. FIG. 7C shows results of a Northernblot analysis of total RNA extracted from 293FT cells transfected withU6 expression constructs carrying long or short tracrRNA, as well asSpCas9 and DR-EMX1(1)-DR. Left and right panels are from 293FT cellstransfected without or with SpRNase III, respectively. U6 indicateloading control blotted with a probe targeting human U6 snRNA.Transfection of the short tracrRNA expression construct led to abundantlevels of the processed form of tracrRNA (˜75 bp). Very low amounts oflong tracrRNA are detected on the Northern blot.

To promote precise transcriptional initiation, the RNA polymeraseIII-based U6 promoter was selected to drive the expression of tracrRNA(FIG. 2C). Similarly, a U6 promoter-based construct was developed toexpress a pre-crRNA array consisting of a single spacer flanked by twodirect repeats (DRs, also encompassed by the term “tracr-matesequences”; FIG. 2C). The initial spacer was designed to target a33-base-pair (bp) target site (30-bp protospacer plus a 3-bp CRISPRmotif (PAM) sequence satisfying the NGG recognition motif of Cas9) inthe human EMX1 locus (FIG. 2C), a key gene in the development of thecerebral cortex.

To test whether heterologous expression of the CRISPR system (SpCas9,SpRNase III, tracrRNA, and pre-crRNA) in mammalian cells can achievetargeted cleavage of mammalian chromosomes, HEK 293FT cells weretransfected with combinations of CRISPR components. Since DSBs inmammalian nuclei are partially repaired by the non-homologous endjoining (NHEJ) pathway, which leads to the formation of indels, theSurveyor assay was used to detect potential cleavage activity at thetarget EMX1 locus (FIG. 8) (see e.g. Guschin et al., 2010, Methods MolBiol 649: 247). Co-transfection of all four CRISPR components was ableto induce up to 5.0% cleavage in the protospacer (see FIG. 2D).Co-transfection of all CRISPR components minus SpRNase III also inducedup to 4.7% indel in the protospacer, suggesting that there may beendogenous mammalian RNases that are capable of assisting with crRNAmaturation, such as for example the related Dicer and Drosha enzymes.Removing any of the remaining three components abolished the genomecleavage activity of the CRISPR system (FIG. 2D). Sanger sequencing ofamplicons containing the target locus verified the cleavage activity: in43 sequenced clones, 5 mutated alleles (11.6%) were found. Similarexperiments using a variety of guide sequences produced indelpercentages as high as 29% (see FIGS. 4-7, 12, and 13). These resultsdefine a three-component system for efficient CRISPR-mediated genomemodification in mammalian cells. To optimize the cleavage efficiency.Applicants also tested whether different isoforms of tracrRNA affectedthe cleavage efficiency and found that, in this example system, only theshort (89-bp) transcript form was able to mediate cleavage of the humanEMX1 genomic locus (FIG. 7B).

FIG. 14 provides an additional Northern blot analysis of crRNAprocessing in mammalian cells. FIG. 14A illustrates a schematic showingthe expression vector for a single spacer flanked by two direct repeats(DR-EMX1(1)-DR). The 30 bp spacer targeting the human EMX1 locusprotospacer 1 (see FIG. 6) and the direct repeat sequences are shown inthe sequence beneath FIG. 14A. The line indicates the region whosereverse-complement sequence was used to generate Northern blot probesfor EMX1(1) crRNA detection. FIG. 14B shows a Northern blot analysis oftotal RNA extracted from 293FT cells transfected with U6 expressionconstructs carrying DR-EMX1(1)-DR. Left and right panels are from 293FTcells transfected without or with SpRNase III respectively.DR-EMX1(1)-DR was processed into mature crRNAs only in the presence ofSpCas9 and short tracrRNA and was not dependent on the presence ofSpRNase III. The mature crRNA detected from transfected 293FT total RNAis ˜33 bp and is shorter than the 39-42 bp mature crRNA from S.pyogenes. These results demonstrate that a CRISPR system can betransplanted into eukaryotic cells and reprogrammed to facilitatecleavage of endogenous mammalian target polynucleotides.

FIG. 2 illustrates the bacterial CRISPR system described in thisexample. FIG. 2A illustrates a schematic showing the CRISPR locus 1 fromStreptococcus pyogenes SF370 and a proposed mechanism of CRISPR-mediatedDNA cleavage by this system. Mature crRNA processed from the directrepeat-spacer array directs Cas9 to genomic targets consisting ofcomplimentary protospacers and a protospacer-adjacent motif (PAM). Upontarget-spacer base pairing, Cas9 mediates a double-strand break in thetarget DNA. FIG. 2B illustrates engineering of S. pyogenes Cas9 (SpCas9)and RNase III (SpRNase III) with nuclear localization signals (NLSs) toenable import into the mammalian nucleus. FIG. 2C illustrates mammalianexpression of SpCas9 and SpRNase III driven by the constitutive EF1apromoter and tracrRNA and pre-crRNA array (DR-Spacer-DR) driven by theRNA Pol3 promoter U6 to promote precise transcription initiation andtermination. A protospacer from the human EMX1 locus with a satisfactoryPAM sequence is used as the spacer in the pre-crRNA array. FIG. 2Dillustrates surveyor nuclease assay for SpCas9-mediated minor insertionsand deletions. SpCas9 was expressed with and without SpRNase III,tracrRNA, and a pre-crRNA array carrying the EMX1-target spacer. FIG. 2Eillustrates a schematic representation of base pairing between targetlocus and EMX1-targeting crRNA, as well as an example chromatogramshowing a micro deletion adjacent to the SpCas9 cleavage site. FIG. 2Fillustrates mutated alleles identified from sequencing analysis of 43clonal amplicons showing a variety of micro insertions and deletions.Dashes indicate deleted bases, and non-aligned or mismatched basesindicate insertions or mutations. Scale bar=10 μm.

To further simplify the three-component system, a chimericcrRNA-tracrRNA hybrid design was adapted, where a mature crRNA(comprising a guide sequence) is fused to a partial tracrRNA via astem-loop to mimic the natural crRNA:tracrRNA duplex (FIG. 3A). Toincrease co-delivery efficiency, a bicistronic expression vector wascreated to drive co-expression of a chimeric RNA and SpCas9 intransfected cells (FIGS. 3A and 8). In parallel, the bicistronic vectorswere used to express a pre-crRNA (DR-guide sequence-DR) with SpCas9, toinduce processing into crRNA with a separately expressed tracrRNA(compare FIG. 13B top and bottom). FIG. 9 provides schematicillustrations of bicistronic expression vectors for pre-crRNA array(FIG. 9A) or chimeric crRNA (represented by the short line downstream ofthe guide sequence insertion site and upstream of the EF1α promoter inFIG. 9B) with hSpCas9, showing location of various elements and thepoint of guide sequence insertion. The expanded sequence around thelocation of the guide sequence insertion site in FIG. 9B also shows apartial DR sequence (GTTTTAGAGCTA (SEQ ID NO: 27)) and a partialtracrRNA sequence (TAGCAAGTTAAAATAAGGCTAGTCCGTTITT (SEQ ID NO: 28)).Guide sequences can be inserted between BbsI sites using annealedoligonucleotides. Sequence design for the oligonucleotides are shownbelow the schematic illustrations in FIG. 9, with appropriate ligationadapters indicated. WPRE represents the Woodchuck hepatitis viruspost-transcriptional regulatory element. The efficiency of chimericRNA-mediated cleavage was tested by targeting the same EMX1 locusdescribed above. Using both Surveyor assay and Sanger sequencing ofamplicons, Applicants confirmed that the chimeric RNA design facilitatescleavage of human EMX1 locus with approximately a 4.7% modification rate(FIG. 4).

Generalizability of CRISPR-mediated cleavage in eukaryotic cells wastested by targeting additional genomic loci in both human and mousecells by designing chimeric RNA targeting multiple sites in the humanEMX1 and PVALB, as well as the mouse Th loci. FIG. 15 illustrates theselection of some additional targeted protospacers in human PVALB (FIG.15A) and mouse Th (FIG. 15B) loci. Schematics of the gene loci and thelocation of three protospacers within the last exon of each areprovided. The underlined sequences include 30 bp of protospacer sequenceand 3 bp at the 3′ end corresponding to the PAM sequences. Protospacerson the sense and anti-sense strands are indicated above and below theDNA sequences, respectively. A modification rate of 6.3% and 0.75% wasachieved for the human PVALB and mouse Th loci respectively,demonstrating the broad applicability of the CRISPR system in modifyingdifferent loci across multiple organisms (FIGS. 3B and 6). Whilecleavage was only detected with one out of three spacers for each locususing the chimeric constructs, all target sequences were cleaved withefficiency of indel production reaching 27% when using the co-expressedpre-crRNA arrangement (FIG. 6).

FIG. 13 provides a further illustration that SpCas9 can be reprogrammedto target multiple genomic loci in mammalian cells. FIG. 13A provides aschematic of the human EMX1 locus showing the location of fiveprotospacers, indicated by the underlined sequences.

FIG. 13B provides a schematic of the pre-crRNA/trcrRNA complex showinghybridization between the direct repeat region of the pre-crRNA andtracrRNA (top), and a schematic of a chimeric RNA design comprising a 20bp guide sequence, and tracr mate and tracr sequences consisting ofpartial direct repeat and tracrRNA sequences hybridized in a hairpinstructure (bottom). Results of a Surveyor assay comparing the efficacyof Cas9-mediated cleavage at five protospacers in the human EMX1 locusis illustrated in FIG. 13C. Each protospacer is targeted using eitherprocessed pre-crRNA/tracrRNA complex (crRNA) or chimeric RNA (chiRNA).

Since the secondary structure of RNA can be crucial for intermolecularinteractions, a structure prediction algorithm based on minimum freeenergy and Boltzmann-weighted structure ensemble was used to compare theputative secondary structure of all guide sequences used in our genometargeting experiment (FIG. 3B) (see e.g. Gruber et al., 2008, NucleicAcids Research, 36: W70). Analysis revealed that in most cases, theeffective guide sequences in the chimeric crRNA context weresubstantially free of secondary structure motifs, whereas theineffective guide sequences were more likely to form internal secondarystructures that could prevent base pairing with the target protospacerDNA. It is thus possible that variability in the spacer secondarystructure might impact the efficiency of CRISPR-mediated interferencewhen using a chimeric crRNA.

FIG. 3 illustrates example expression vectors. FIG. 3A provides aschematic of a bi-cistronic vector for driving the expression of asynthetic crRNA-tracrRNA chimera (chimeric RNA) as well as SpCas9. Thechimeric guide RNA contains a 20-bp guide sequence corresponding to theprotospacer in the genomic target site. FIG. 3B provides a schematicshowing guide sequences targeting the human EMX1, PVALB, and mouse Thloci, as well as their predicted secondary structures. The modificationefficiency at each target site is indicated below the RNA secondarystructure drawing (EMX1, n=216 amplicon sequencing reads; PVALB, n=224reads; Th, n=265 reads). The folding algorithm produced an output witheach base colored according to its probability of assuming the predictedsecondary structure, as indicated by a rainbow scale that is reproducedin FIG. 3B in gray scale. Further vector designs for SpCas9 are shown inFIG. 44, which illustrates single expression vectors incorporating a U6promoter linked to an insertion site for a guide oligo, and a Cbhpromoter linked to SpCas9 coding sequence. The vector shown in FIG. 44bincludes a tracrRNA coding sequence linked to an H1 promoter.

To test whether spacers containing secondary structures are able tofunction in prokaryotic cells where CRISPRs naturally operate,transformation interference of protospacer-bearing plasmids were testedin an E. coli strain heterologously expressing the S. pyogenes SF370CRISPR locus 1 (FIG. 10). The CRISPR locus was cloned into a low-copy E.coli expression vector and the crRNA array was replaced with a singlespacer flanked by a pair of DRs (pCRISPR). E. coli strains harboringdifferent pCRISPR plasmids were transformed with challenge plasmidscontaining the corresponding protospacer and PAM sequences (FIG. 10C).In the bacterial assay, all spacers facilitated efficient CRISPRinterference (FIG. 4C). These results suggest that there may beadditional factors affecting the efficiency of CRISPR activity inmammalian cells.

To investigate the specificity of CRISPR-mediated cleavage, the effectof single-nucleotide mutations in the guide sequence on protospacercleavage in the mammalian genome was analyzed using a series ofEMX1-targeting chimeric crRNAs with single point mutations (FIG. 4A).FIG. 4B illustrates results of a Surveyor nuclease assay comparing thecleavage efficiency of Cas9 when paired with different mutant chimericRNAs. Single-base mismatch up to 12-bp 5′ of the PAM substantiallyabrogated genomic cleavage by SpCas9, whereas spacers with mutations atfarther upstream positions retained activity against the originalprotospacer target (FIG. 4B). In addition to the PAM, SpCas9 hassingle-base specificity within the last 12-bp of the spacer.Furthermore, CRISPR is able to mediate genomic cleavage as efficientlyas a pair of TALE nucleases (TALEN) targeting the same EMX1 protospacer.FIG. 4C provides a schematic showing the design of TALENs targetingEMX1, and FIG. 4D shows a Surveyor gel comparing the efficiency of TALENand Cas9 (n=3).

Having established a set of components for achieving CRISPR-mediatedgene editing in mammalian cells through the error-prone NHEJ mechanism,the ability of CRISPR to stimulate homologous recombination (HR), a highfidelity gene repair pathway for making precise edits in the genome, wastested. The wild type SpCas9 is able to mediate site-specific DSBs,which can be repaired through both NHEJ and HR. In addition, anaspartate-to-alanine substitution (D10A) in the RuvC I catalytic domainof SpCas9 was engineered to convert the nuclease into a nickase(SpCas9n; illustrated in FIG. 5A) (see e.g. Sapranauskas et al., 2011,Nucleic Acids Research, 39: 9275; Gasiunas et al., 2012, Proc. Natl.Acad. Sci. USA, 109:E2579), such that nicked genomic DNA undergoes thehigh-fidelity homology-directed repair (HDR). Surveyor assay confirmedthat SpCas9n does not generate indels at the EMX1 protospacer target. Asillustrated in FIG. 5B, co-expression of EMX1-targeting chimeric crRNAwith SpCas9 produced indels in the target site, whereas co-expressionwith SpCas9n did not (n=3). Moreover, sequencing of 327 amplicons didnot detect any indels induced by SpCas9n. The same locus was selected totest CRISPR-mediated HR by co-transfecting HEK 293FT cells with thechimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, as well as a HRtemplate to introduce a pair of restriction sites (HindIII and NheI)near the protospacer. FIG. 5C provides a schematic illustration of theHR strategy, with relative locations of recombination points and primerannealing sequences (arrows). SpCas9 and SpCas9n indeed catalyzedintegration of the HR template into the EMX1 locus. PCR amplification ofthe target region followed by restriction digest with HindIII revealedcleavage products corresponding to expected fragment sizes (arrows inrestriction fragment length polymorphism gel analysis shown in FIG. 5D),with SpCas9 and SpCas9n mediating similar levels of HR efficiencies.Applicants further verified HR using Sanger sequencing of genomicamplicons (FIG. 5E). These results demonstrate the utility of CRISPR forfacilitating targeted gene insertion in the mammalian genome. Given the14-bp (12-bp from the spacer and 2-bp from the PAM) target specificityof the wild type SpCas9, the availability of a nickase can significantlyreduce the likelihood of off-target modifications, since single strandbreaks are not substrates for the error-prone NHEJ pathway.

Expression constructs mimicking the natural architecture of CRISPR lociwith arrayed spacers (FIG. 2A) were constructed to test the possibilityof multiplexed sequence targeting. Using a single CRISPR array encodinga pair of EMX1- and PVALB-targeting spacers, efficient cleavage at bothloci was detected (FIG. 4F, showing both a schematic design of the crRNAarray and a Surveyor blot showing efficient mediation of cleavage).Targeted deletion of larger genomic regions through concurrent DSBsusing spacers against two targets within EMX1 spaced by 119 bp was alsotested, and a 1.6% deletion efficacy (3 out of 182 amplicons; FIG. 4G)was detected. This demonstrates that the CRISPR system can mediatemultiplexed editing within a single genome.

Example 2 CRISPR System Modifications and Alternatives

The ability to use RNA to program sequence-specific DNA cleavage definesa new class of genome engineering tools for a variety of research andindustrial applications. Several aspects of the CRISPR system can befurther improved to increase the efficiency and versatility of CRISPRtargeting. Optimal Cas9 activity may depend on the availability of freeMg²⁺ at levels higher than that present in the mammalian nucleus (seee.g. Jinek et al., 2012, Science, 337:816), and the preference for anNGG motif immediately downstream of the protospacer restricts theability to target on average every 12-bp in the human genome (FIG. 11,evaluating both plus and minus strands of human chromosomal sequences).Some of these constraints can be overcome by exploring the diversity ofCRISPR loci across the microbial metagenome (see e.g. Makarova et al.,2011, Nat Rev Microbiol, 9:467). Other CRISPR loci may be transplantedinto the mammalian cellular milieu by a process similar to thatdescribed in Example 1. For example, FIG. 12 illustrates adaptation ofthe Type II CRISPR system from CRISPR 1 of Streptococcus thermophilusLMD-9 for heterologous expression in mammalian cells to achieveCRISPR-mediated genome editing. FIG. 12A provides a Schematicillustration of CRISPR 1 from S. thermophilus LMD-9. FIG. 12Billustrates the design of an expression system for the S. thermophilusCRISPR system. Human codon-optimized hStCas9 is expressed using aconstitutive EF1α promoter. Mature versions of tracrRNA and crRNA areexpressed using the U6 promoter to promote precise transcriptioninitiation. Sequences from the mature crRNA and tracrRNA areillustrated. A single base indicated by the lower case “a” in the crRNAsequence is used to remove the polyU sequence, which serves as a RNApolIII transcriptional terminator. FIG. 12C provides a schematic showingguide sequences targeting the human EMX1 locus as well as theirpredicted secondary structures. The modification efficiency at eachtarget site is indicated below the RNA secondary structures. Thealgorithm generating the structures colors each base according to itsprobability of assuming the predicted secondary structure, which isindicated by a rainbow scale reproduced in FIG. 12C in gray scale. FIG.12D shows the results of hStCas9-mediated cleavage in the target locususing the Surveyor assay. RNA guide spacers 1 and 2 induced 14% and6.4%, respectively. Statistical analysis of cleavage activity acrossbiological replica at these two protospacer sites is also provided inFIG. 6. FIG. 16 provides a schematic of additional protospacer andcorresponding PAM sequence targets of the S. thermophilus CRISPR systemin the human EMX1 locus. Two protospacer sequences are highlighted andtheir corresponding PAM sequences satisfying NNAGAAW motif are indicatedby underlining 3′ with respect to the corresponding highlightedsequence. Both protospacers target the anti-sense strand.

Example 3 Sample Target Sequence Selection Algorithm

A software program is designed to identify candidate CRISPR targetsequences on both strands of an input DNA sequence based on desiredguide sequence length and a CRISPR motif sequence (PAM) for a specifiedCRISPR enzyme. For example, target sites for Cas9 from S. pyogenes, withPAM sequences NGG, may be identified by searching for 5′-N_(x)-NGG-3′both on the input sequence and on the reverse-complement of the input.Likewise, target sites for Cas9 of S. thermophilus CRISPR1, with PAMsequence NNAGAAW, may be identified by searching for 5′-N_(x)-NNAGAAW-3′(SEQ ID NO: 29) both on the input sequence and on the reverse-complementof the input. Likewise, target sites for Cas9 of S. thermophilusCRISPR3, with PAM sequence NGGNG, may be identified by searching for5′-N_(x)-NGGNG-3′ both on the input sequence and on thereverse-complement of the input. The value “x” in N_(x) may be fixed bythe program or specified by the user, such as 20.

Since multiple occurrences in the genome of the DNA target site may leadto nonspecific genome editing, after identifying all potential sites,the program filters out sequences based on the number of times theyappear in the relevant reference genome. For those CRISPR enzymes forwhich sequence specificity is determined by a ‘seed’ sequence, such asthe 11-12 bp 5′ from the PAM sequence, including the PAM sequenceitself, the filtering step may be based on the seed sequence. Thus, toavoid editing at additional genomic loci, results are filtered based onthe number of occurrences of the seed:PAM sequence in the relevantgenome. The user may be allowed to choose the length of the seedsequence. The user may also be allowed to specify the number ofoccurrences of the seed:PAM sequence in a genome for purposes of passingthe filter. The default is to screen for unique sequences. Filtrationlevel is altered by changing both the length of the seed sequence andthe number of occurrences of the sequence in the genome. The program mayin addition or alternatively provide the sequence of a guide sequencecomplementary to the reported target sequence(s) by providing thereverse complement of the identified target sequence(s).

Further details of methods and algorithms to optimize sequence selectioncan be found in U.S. application Ser. No. 61/836,080 (attorney docket44790.11.2022); incorporated herein by reference.

Example 4 Evaluation of Multiple Chimeric crRNA-tracrRNA Hybrids

This example describes results obtained for chimeric RNAs (chiRNAs;comprising a guide sequence, a tracr mate sequence, and a tracr sequencein a single transcript) having tracr sequences that incorporatedifferent lengths of wild-type tracrRNA sequence. FIG. 18a illustrates aschematic of a bicistronic expression vector for chimeric RNA and Cas9.Cas9 is driven by the CBh promoter and the chimeric RNA is driven by aU6 promoter. The chimeric guide RNA consists of a 20 bp guide sequence(Ns) joined to the tracr sequence (running from the first “U” of thelower strand to the end of the transcript), which is truncated atvarious positions as indicated. The guide and tracr sequences areseparated by the tracr-mate sequence GUUUUAGAGCUA (SEQ ID NO: 30)followed by the loop sequence GAAA. Results of SURVEYOR assays forCas9-mediated indels at the human EMX1 and PVALB loci are illustrated inFIGS. 18b and 18c , respectively. Arrows indicate the expected SURVEYORfragments. ChiRNAs are indicated by their “+n” designation, and crRNArefers to a hybrid RNA where guide and tracr sequences are expressed asseparate transcripts. Quantification of these results, performed intriplicate, are illustrated by histogram in FIGS. 19a and 19b ,corresponding to FIGS. 18b and 18c , respectively (“N.D.” indicates noindels detected). Protospacer IDs and their corresponding genomictarget, protospacer sequence, PAM sequence, and strand location areprovided in Table D. Guide sequences were designed to be complementaryto the entire protospacer sequence in the case of separate transcriptsin the hybrid system, or only to the underlined portion in the case ofchimeric RNAs.

TABLE D protospacer genomic ID target protospacer sequence (5′ to 3′)PAM strand 1 EMX1 GGACATCGATGTCACCTCCAATGACTAG TGG + GG (SEQ ID NO: 31)2 EMX1 CATTGGAGGTGACATCGATGTCCTCCCC TGG − AT (SEQ ID NO: 32) 3 EMX1GGAAGGGCCTGAGTCCGAGCAGAAGAA GGG + GAA (SEQ ID NO: 33) 4 PVALBGGTGGCGAGAGGGGCCGAGATTGGGTGT AGG + TC (SEQ ID NO: 34) 5 PVALBATGCAGGAGGGTGGCGAGAGGGGCCGA TGG + GAT (SEQ ID NO: 35)

Cell Culture and Transfection

Human embryonic kidney (HEK) cell line 293FT (Life Technologies) wasmaintained in Dulbecco's modified Eagle's Medium (DMEM) supplementedwith 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (LifeTechnologies), 100U/mL penicillin, and 100 μg/mL streptomycin at 37° C.with 5% CO₂ incubation. 293FT cells were seeded onto 24-well plates(Corning) 24 hours prior to transfection at a density of 150,000 cellsper well. Cells were transfected using Lipofectamine 2000 (LifeTechnologies) following the manufacturer's recommended protocol. Foreach well of a 24-well plate, a total of 500 ng plasmid was used.

SURVEYOR Assay for Genome Modification

293FT cells were transfected with plasmid DNA as described above. Cellswere incubated at 37° C. for 72 hours post-transfection prior to genomicDNA extraction. Genomic DNA was extracted using the QuickExtract DNAExtraction Solution (Epicentre) following the manufacturer's protocol.Briefly, pelleted cells were resuspended in QuickExtract solution andincubated at 65° C. for 15 minutes and 98° C. for 10 minutes. Thegenomic region flanking the CRISPR target site for each gene was PCRamplified (primers listed in Table E), and products were purified usingQiaQuick Spin Column (Qiagen) following the manufacturer's protocol. 400ng total of the purified PCR products were mixed with 2 μl 10× Taq DNAPolymerase PCR buffer (Enzymatics) and ultrapure water to a final volumeof 201, and subjected to a re-annealing process to enable heteroduplexformation: 95° C. for 10 min, 95° C. to 85° C. ramping at −2° C./s, 85°C. to 25° C. at −0.25° C./s, and 25° C. hold for 1 minute. Afterre-annealing, products were treated with SURVEYOR nuclease and SURVEYORenhancer S (Transgenomics) following the manufacturer's recommendedprotocol, and analyzed on 4-20% Novex TBE poly-acrylamide gels (LifeTechnologies). Gels were stained with SYBR Gold DNA stain (LifeTechnologies) for 30 minutes and imaged with a Gel Doc gel imagingsystem (Bio-rad). Quantification was based on relative band intensities.

TABLE E genomic primer sequence primer name target (5′ to 3′) Sp-EMX1-FEMX1 AAAACCACCCTTCTCTCTGGC (SEQ ID NO: 36) Sp-EMX1-R EMX1GGAGATTGGAGACACGGAGA G (SEQ ID NO: 37) Sp-PVALB-F PVALBCTGGAAAGCCAATGCCTGAC (SEQ ID NO: 38) Sp-PVALB-R PVALBGGCAGCAAACTCCTTGTCCT (SEQ ID NO: 39)

Computational Identification of Unique CRISPR Target Sites

To identify unique target sites for the S. pyogenes SF370 Cas9 (SpCas9)enzyme in the human, mouse, rat, zebrafish, fruit fly, and C. elegansgenome, we developed a software package to scan both strands of a DNAsequence and identify all possible SpCas9 target sites. For thisexample, each SpCas9 target site was operationally defined as a 20 bpsequence followed by an NGG protospacer adjacent motif (PAM) sequence,and we identified all sequences satisfying this 5′-N₂₀-NGG-3′ definitionon all chromosomes. To prevent non-specific genome editing, afteridentifying all potential sites, all target sites were filtered based onthe number of times they appear in the relevant reference genome. Totake advantage of sequence specificity of Cas9 activity conferred by a‘seed’ sequence, which can be, for example, approximately 11-12 bpsequence 5′ from the PAM sequence, 5′-NNNNNNNNNN-NGG-3′ sequences wereselected to be unique in the relevant genome. All genomic sequences weredownloaded from the UCSC Genome Browser (Human genome hg19. Mouse genomemm9, Rat genome rn5, Zebrafish genome danRer7, D. melanogaster genomedm4 and C. elegans genome ce10). The full search results are availableto browse using UCSC Genome Browser information. An examplevisualization of some target sites in the human genome is provided inFIG. 21.

Initially, three sites within the EMX1 locus in human HEK 293FT cellswere targeted. Genome modification efficiency of each chiRNA wasassessed using the SURVEYOR nuclease assay, which detects mutationsresulting from DNA double-strand breaks (DSBs) and their subsequentrepair by the non-homologous end joining (NHEJ) DNA damage repairpathway. Constructs designated chiRNA(+n) indicate that up to the +nnucleotide of wild-type tracrRNA is included in the chimeric RNAconstruct, with values of 48, 54, 67, and 85 used for n. Chimeric RNAscontaining longer fragments of wild-type tracrRNA (chiRNA(+67) andchiRNA(+85)) mediated DNA cleavage at all three EMX1 target sites, withchiRNA(+85) in particular demonstrating significantly higher levels ofDNA cleavage than the corresponding crRNA/tracrRNA hybrids thatexpressed guide and tracr sequences in separate transcripts (FIGS. 18band 19a ). Two sites in the PVALB locus that yielded no detectablecleavage using the hybrid system (guide sequence and tracr sequenceexpressed as separate transcripts) were also targeted using chiRNAs.chiRNA(+67) and chiRNA(+85) were able to mediate significant cleavage atthe two PVALB protospacers (FIGS. 18c and 19b ).

For all five targets in the EMX1 and PVALB loci, a consistent increasein genome modification efficiency with increasing tracr sequence lengthwas observed. Without wishing to be bound by any theory, the secondarystructure formed by the 3′ end of the tracrRNA may play a role inenhancing the rate of CRISPR complex formation. An illustration ofpredicted secondary structures for each of the chimeric RNAs used inthis example is provided in FIG. 21. The secondary structure waspredicted using RNAfold using minimum free energy and partition functionalgorithm. Pseudocolor for each based (reproduced in grayscale)indicates the probability of pairing. Because chiRNAs with longer tracrsequences were able to cleave targets that were not cleaved by nativeCRISPR crRNA/tracrRNA hybrids, it is possible that chimeric RNA may beloaded onto Cas9 more efficiently than its native hybrid counterpart. Tofacilitate the application of Cas9 for site-specific genome editing ineukaryotic cells and organisms, all predicted unique target sites forthe S. pyogenes Cas9 were computationally identified in the human,mouse, rat, zebra fish, C. elegans, and D. melanogaster genomes.Chimeric RNAs can be designed for Cas9 enzymes from other microbes toexpand the target space of CRISPR RNA-programmable nucleases.

FIG. 22 illustrates an exemplary bicistronic expression vector forexpression of chimeric RNA including up to the +85 nucleotide ofwild-type tracr RNA sequence, and SpCas9 with nuclear localizationsequences. SpCas9 is expressed from a CBh promoter and terminated withthe bGH polyA signal (bGH pA). The expanded sequence illustratedimmediately below the schematic corresponds to the region surroundingthe guide sequence insertion site, and includes, from 5′ to 3′,3′-portion of the U6 promoter (first shaded region), BbsI cleavage sites(arrows), partial direct repeat (tracr mate sequence GTTTTAGAGCTA (SEQID NO: 27), underlined), loop sequence GAAA, and +85 tracr sequence(underlined sequence following loop sequence). An exemplary guidesequence insert is illustrated below the guide sequence insertion site,with nucleotides of the guide sequence for a selected target representedby an “N”.

Sequences described in the above examples are as follows (polynucleotidesequences are 5′ to 3′):

U6-short tracrRNA (Streptococcus pyogenes SF370):GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC TTTTTTT (SEQ ID NO: 40) (bold = tracrRNA sequence; underline =terminator sequence) U6-long tracrRNA (Streptococcus pyogenes SF370):GAGGGCCCATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGTAGTATTAAGTATTGTTTTATGGCTGATAAATTTCTTTGAATTTCTCCTTGATTATTTGTTATAAAAGTTATAAAATAATCTTGTTGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 41)U6-DR-BbsI backbone-DR (Streptococcus pyogenes SF370):GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACGGGTCTTCGAGAAGACGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC (SEQ ID NO: 42)U6-chimeric RNA-BbsI backbone (Streptococcus pyogenes SF370)GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG (SEQ ID NO: 43) NLS-SpCas9-EGFP:MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 44)SpCas9-EGFP-NLS: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKRPAATKKAGQAKKKK (SEQ ID NO: 45) NLS-SpCas9-EGFP-NLS:MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKRPAATKKAGQAKKKK (SEQ ID NO: 46) NLS-SpCas9-NLS:MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK (SEQ ID NO: 47) NLS-mCherry-SpRNase3:MFLFLSLTSFLSSSRTLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGSKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFDAYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMIPQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVSIVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEV (SEQ ID NO: 48) SpRNase3-mCherry-NLS:MKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFDAYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMIPQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVSIVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEVGSVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKKRPAATKKAGQAKKKK (SEQ ID NO: 49)NLS-SpCas9n-NLS (the D10A nickase mutation is lowercase):MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLaIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK (SEQ ID NO: 50) hEMX1-HR Template-HindII-NheI:GAATGCTGCCCTCAGACCCGCTTCCTCCCTGTCCTTGTCTGTCCAAGGAGAATGAGGTCTCACTGGTGGATTTCGGACTACCCTGAGGAGCTGGCACCTGAGGGACAAGGCCCCCCACCTGCCCAGCTCCAGCCTCTGATGAGGGGTGGGAGAGAGCTACATGAGGTTGCTAAGAAAGCCTCCCCTGAAGGAGACCACACAGTGTGTGAGGTTGGAGTCTCTAGCAGCGGGTTCTGTGCCCCCAGGGATAGTCTGGCTGTCCAGGCACTGCTCTTGATATAAACACCACCTCCTAGTTATGAAACCATGCCCATTCTGCCTCTCTGTATGGAAAAGAGCATGGGGCTGGCCCGTGGGGTGGTGTCCACTTTAGGCCCTGTGGGAGATCATGGGAACCCACGCAGTGGGTCATAGGCTCTCTCATTTACTACTCACATCCACTCTGTGAAGAAGCGATTATGATCTCTCCTCTAGAAACTCGTAGAGTCCCATGTCTGCCGGCTTCCAGAGCCTGCACTCCTCCACCTTGGCTTGGCTTTGCTGGGGCTAGAGGAGCTAGGATGCACAGCAGCTCTGTGACCCTTTGTTTGAGAGGAACAGGAAAACCACCCTTCTCTCTGGCCCACTGTGTCCTCTTCCTGCCCTGCCATCCCCTTCTGTGAATGTTAGACCCATGGGAGCAGCTGGTCAGAGGGGACCCCGGCCTGGGGCCCCTAACCCTATGTAGCCTCAGTCTTCCCATCAGGCTCTCAGCTCAGCCTGAGTGTTGAGGCCCCAGTGGCTGCTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACaagcttgctagcGGTGGGCAACCACAAACCCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAGTCATGGCCCCACAGGGCTTGAAGCCCGGGGCCGCCATTGACAGAGGGACAAGCAATGGGCTGGCTGAGGCCTGGGACCACTTGGCCTTCTCCTCGGAGAGCCTGCCTGCCTGGGCGGGCCCGCCCGCCACCGCAGCCTCCCAGCTGCTCTCCGTGTCTCCAATCTCCCTTTTGTTTTGATGCATTTCTGTTTTAATTTATTTTCCAGGCACCACTGTAGTTTAGTGATCCCCAGTGTCCCCCTTCCCTATGGGAATAATAAAAGTCTCTCTCTTAATGACACGGGCATCCAGCTCCAGCCCCAGAGCCTGGGGTGGTAGATTCCGGCTCTGAGGGCCAGTGGGGGCTGGTAGAGCAAACGCGTTCAGGGCCTGGGAGCCTGGGGTGGGGTACTGGTGGAGGGGGTCAAGGGTAATTCATTAACTCCTCTCTTTTGTTGGGGGACCCTGGTCTCTACCTCCAGCTCCACAGCAGGAGAAACAGGCTAGACATAGGGAAGGGCCATCCTGTATCTTGAGGGAGGACAGGCCCAGGTCTTTCTTAACGTATTGAGAGGTGGGAATCAGGCCCAGGTAGTTCAATGGGAGAGGGAGAGTGCTTCCCTCTGCCTAGAGACTCTGGTGGCTTCTCCAGTTGAGGAGAAACCAGAGGAAAGGGGAGGATTGGGGTCTGGGGGAGGGAACACCATTCACAAAGGCTGACGGTTCCAGTCCGAAGTCGTGGGCCCACCAGGATGCTCACCTGTCCTTGGAGAACCGCTGGGCAGGTTGAGACTGCAGAGACAGGGCTTAAGGCTGAGCCTGCAACCAGTCCCCAGTGACTCAGGGCCTCCTCAGCCCAAGAAAGAGCAACGTGCCAGGGCCCGCTGAGCTCTTGTGTTCACCTG (SEQ ID NO: 51) NLS-StCsn1-NLS:MKRPAATKKAGQAKKKKSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDFKRPAATKKAGQAKKKK (SEQ ID NO: 52) U6-St_tracrRNA(7-97):GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTTACTTAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAA (SEQ ID NO: 53) U6-DR-spacer-DR (S. pyogenes SF370)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgggttttagagctatgctgttttgaatggtcccaaaacNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNgttttagagctatgctgttttgaatggtcccaaaacT TTTTTT (SEQ ID NO: 54) (lowercase underline = direct repeat; N =guide sequence; bold = terminator)Chimeric RNA containing +48 tracr RNA (S. pyogenes SF370)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaataaggctagtccgTTTTTTT (SEQ ID NO: 55) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator) Chimeric RNA containing +54 tracr RNA (S. pyogenes SF370)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaTTTTTTTT (SEQ ID NO: 56) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator) Chimeric RNA containing +67 tracr RNA (S. pyogenes SF370)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT (SEQ ID NO: 57) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator) Chimeric RNA containing +85 tracr RNA (S. pyogenes SF370)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTT (SEQ ID NO: 58) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator) CBh-NLS-SpCas9-NLSCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGaccggtgccaccATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACTTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTAGCAGCAGGACGCTTTAA(SEQ ID NO: 59) (underline = NLS-hSpCas9-NLS)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 21)(N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 22) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgt TTTTTT (SEQ ID NO: 23) (N =guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 60) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 61) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgt TTTTTT (SEQ ID NO: 62) (N =guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaaatcttgcagaagctacaatgataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 63) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagctacaatgataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 64) (N = guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagctacaatgataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgt TTTTTT (SEQ ID NO: 65) (N =guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Example chimeric RNA for S. thermophilus LMD-9 CRISPR3 Cas9 (with PAM ofNGGNG) NNNNNNNNNNNNNNNNNNNNgttttagagctgtgGAAAcacagcgagttaaaataaggcttagtccgtactcaacttgaaaaggtggcaccgattcggtgt TTTTTT (SEQ ID NO: 66) (N =guide sequence; first underline =tracr mate sequence; second underline = tracr sequence; bold =terminator)Codon-optimized version of Cas9 from S. thermophilus LMD-9 CRISPR3 locus(with an NLS at both 5′ and 3′ ends)ATGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGACCAAGCCCTACAGCATCGGCCTGGACATCGGCACCAATAGCGTGGGCTGGGCCGTGACCACCGACAACTACAAGGTGCCCAGCAAGAAAATGAAGGTGCTGGGCAACACCTCCAAGAAGTACATCAAGAAAAACCTGCTGGGCGTGCTGCTGTTCGACAGCGGCATTACAGCCGAGGGCAGACGGCTGAAGAGAACCGCCAGACGGCGGTACACCCGGCGGAGAAACAGAATCCTGTATCTGCAAGAGATCTTCAGCACCGAGATGGCTACCCTGGACGACGCCTTCTTCCAGCGGCTGGACGACAGCTTCCTGGTGCCCGACGACAAGCGGGACAGCAAGTACCCCATCTTCGGCAACCTGGTGGAAGAGAAGGCCTACCACGACGAGTTCCCCACCATCTACCACCTGAGAAAGTACCTGGCCGACAGCACCAAGAAGGCCGACCTGAGACTGGTGTATCTGGCCCTGGCCCACATGATCAAGTACCGGGGCCACTTCCTGATCGAGGGCGAGTTCAACAGCAAGAACAACGACATCCAGAAGAACTTCCAGGACTTCCTGGACACCTACAACGCCATCTTCGAGAGCGACCTGTCCCTGGAAAACAGCAAGCAGCTGGAAGAGATCGTGAAGGACAAGATCAGCAAGCTGGAAAAGAAGGACCGCATCCTGAAGCTGTTCCCCGGCGAGAAGAACAGCGGAATCTTCAGCGAGTTTCTGAAGCTGATCGTGGGCAACCAGGCCGACTTCAGAAAGTGCTTCAACCTGGACGAGAAAGCCAGCCTGCACTTCAGCAAAGAGAGCTACGACGAGGACCTGGAAACCCTGCTGGGATATATCGGCGACGACTACAGCGACGTGTTCCTGAAGGCCAAGAAGCTGTACGACGCTATCCTGCTGAGCGGCTTCCTGACCGTGACCGACAACGAGACAGAGGCCCCACTGAGCAGCGCCATGATTAAGCGGTACAACGAGCACAAAGAGGATCTGGCTCTGCTGAAAGAGTACATCCGGAACATCAGCCTGAAAACCTACAATGAGGTGTTCAAGGACGACACCAAGAACGGCTACGCCGGCTACATCGACGGCAAGACCAACCAGGAAGATTTCTATGTGTACCTGAAGAAGCTGCTGGCCGAGTTCGAGGGGGCCGACTACTTTCTGGAAAAAATCGACCGCGAGGATTTCCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCTACCAGATCCATCTGCAGGAAATGCGGGCCATCCTGGACAAGCAGGCCAAGTTCTACCCATTCCTGGCCAAGAACAAAGAGCGGATCGAGAAGATCCTGACCTTCCGCATCCCTTACTACGTGGGCCCCCTGGCCAGAGGCAACAGCGATTTTGCCTGGTCCATCCGGAAGCGCAATGAGAAGATCACCCCCTGGAACTTCGAGGACGTGATCGACAAAGAGTCCAGCGCCGAGGCCTTCATCAACCGGATGACCAGCTTCGACCTGTACCTGCCCGAGGAAAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGACATTCAATGTGTATAACGAGCTGACCAAAGTGCGGTTTATCGCCGAGTCTATGCGGGACTACCAGTTCCTGGACTCCAAGCAGAAAAAGGACATCGTGCGGCTGTACTTCAAGGACAAGCGGAAAGTGACCGATAAGGACATCATCGAGTACCTGCACGCCATCTACGGCTACGATGGCATCGAGCTGAAGGGCATCGAGAAGCAGTTCAACTCCAGCCTGAGCACATACCACGACCTGCTGAACATTATCAACGACAAAGAATTTCTGGACGACTCCAGCAACGAGGCCATCATCGAAGAGATCATCCACACCCTGACCATCTTTGAGGACCGCGAGATGATCAAGCAGCGGCTGAGCAAGTTCGAGAACATCTTCGACAAGAGCGTGCTGAAAAAGCTGAGCAGACGGCACTACACCGGCTGGGGCAAGCTGAGCGCCAAGCTGATCAACGGCATCCGGGACGAGAAGTCCGGCAACACAATCCTGGACTACCTGATCGACGACGGCATCAGCAACCGGAACTTCATGCAGCTGATCCACGACGACGCCCTGAGCTTCAAGAAGAAGATCCAGAAGGCCCAGATCATCGGGGACGAGGACAAGGGCAACATCAAAGAAGTCGTGAAGTCCCTGCCCGGCAGCCCCGCCATCAAGAAGGGAATCCTGCAGAGCATCAAGATCGTGGACGAGCTCGTGAAAGTGATGGGCGGCAGAAAGCCCGAGAGCATCGTGGTGGAAATGGCTAGAGAGAACCAGTACACCAATCAGGGCAAGAGCAACAGCCAGCAGAGACTGAAGAGACTGGAAAAGTCCCTGAAAGAGCTGGGCAGCAAGATTCTGAAAGAGAATATCCCTGCCAAGCTGTCCAAGATCGACAACAACGCCCTGCAGAACGACCGGCTGTACCTGTACTACCTGCAGAATGGCAAGGACATGTATACAGGCGACGACCTGGATATCGACCGCCTGAGCAACTACGACATCGACCATATTATCCCCCAGGCCTTCCTGAAAGACAACAGCATTGACAACAAAGTGCTGGTGTCCTCCGCCAGCAACCGCGGCAAGTCCGATGATGTGCCCAGCCTGGAAGTCGTGAAAAAGAGAAAGACCTTCTGGTATCAGCTGCTGAAAAGCAAGCTGATTAGCCAGAGGAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCCCTGAAGATAAGGCCGGCTTCATCCAGAGACAGCTGGTGGAAACCCGGCAGATCACCAAGCACGTGGCCAGACTGCTGGATGAGAAGTTTAACAACAAGAAGGACGAGAACAACCGGGCCGTGCGGACCGTGAAGATCATCACCCTGAAGTCCACCCTGGTGTCCCAGTTCCGGAAGGACTTCGAGCTGTATAAAGTGCGCGAGATCAATGACTTTCACCACGCCCACGACGCCTACCTGAATGCCGTGGTGGCTTCCGCCCTGCTGAAGAAGTACCCTAAGCTGGAACCCGAGTTCGTGTACGGCGACTACCCCAAGTACAACTCCTTCAGAGAGCGGAAGTCCGCCACCGAGAAGGTGTACTTCTACTCCAACATCATGAATATCTTTAAGAAGTCCATCTCCCTGGCCGATGGCAGAGTGATCGAGCGGCCCCTGATCGAAGTGAACGAAGAGACAGGCGAGAGCGTGTGGAACAAAGAAAGCGACCTGGCCACCGTGCGGCGGGTGCTGAGTTATCCTCAAGTGAATGTCGTGAAGAAGGTGGAAGAACAGAACCACGGCCTGGATCGGGGCAAGCCCAAGGGCCTGTTCAACGCCAACCTGTCCAGCAAGCCTAAGCCCAACTCCAACGAGAATCTCGTGGGGGCCAAAGAGTACCTGGACCCTAAGAAGTACGGCGGATACGCCGGCATCTCCAATAGCTTCACCGTGCTCGTGAAGGGCACAATCGAGAAGGGCGCTAAGAAAAAGATCACAAACGTGCTGGAATTTCAGGGGATCTCTATCCTGGACCGGATCAACTACCGGAAGGATAAGCTGAACTTTCTGCTGGAAAAAGGCTACAAGGACATTGAGCTGATTATCGAGCTGCCTAAGTACTCCCTGTTCGAACTGAGCGACGGCTCCAGACGGATGCTGGCCTCCATCCTGTCCACCAACAACAAGCGGGGCGAGATCCACAAGGGAAACCAGATCTTCCTGAGCCAGAAATTTGTGAAACTGCTGTACCACGCCAAGCGGATCTCCAACACCATCAATGAGAACCACCGGAAATACGTGGAAAACCACAAGAAAGAGTTTGAGGAACTGTTCTACTACATCCTGGAGTTCAACGAGAACTATGTGGGAGCCAAGAAGAACGGCAAACTGCTGAACTCCGCCTTCCAGAGCTGGCAGAACCACAGCATCGACGAGCTGTGCAGCTCCTTCATCGGCCCTACCGGCAGCGAGCGGAAGGGACTGTTTGAGCTGACCTCCAGAGGCTCTGCCGCCGACTTTGAGTTCCTGGGAGTGAAGATCCCCCGGTACAGAGACTACACCCCCTCTAGTCTGCTGAAGGACGCCACCCTGATCCACCAGAGCGTGACCGGCCTGTACGAAACCCGGATCGACCTGGCTAAGCTGGGCGAGGGAAAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAATAA (SEQ ID NO: 67)

Example 5 RNA-Guided Editing of Bacterial Genomes Using CRISPR-CasSystems

Applicants used the CRISPR-associated endonuclease Cas9 to introduceprecise mutations in the genomes of Streptococcus pneumoniae andEscherichia coli. The approach relied on Cas9-directed cleavage at thetargeted site to kill unmutated cells and circumvented the need forselectable markers or counter-selection systems. Cas9 specificity wasreprogrammed by changing the sequence of short CRISPR RNA (crRNA) tomake single- and multi-nucleotide changes carried on editing templates.Simultaneous use of two crRNAs enabled multiplex mutagenesis. In S.pneumoniae, nearly 100% of cells that survived Cas9 cleavage containedthe desired mutation, and 65% when used in combination withrecombineering in E. coli. Applicants exhaustively analyzed Cas9 targetrequirements to define the range of targetable sequences and showedstrategies for editing sites that do not meet these requirements,suggesting the versatility of this technique for bacterial genomeengineering.

The understanding of gene function depends on the possibility ofaltering DNA sequences within the cell in a controlled fashion.Site-specific mutagenesis in eukaryotes is achieved by the use ofsequence-specific nucleases that promote homologous recombination of atemplate DNA containing the mutation of interest. Zinc finger nucleases(ZFNs), transcription activator-like effector nucleases (TALENs) andhoming meganucleases can be programmed to cleave genomes in specificlocations, but these approaches require engineering of new enzymes foreach target sequence. In prokaryotic organisms, mutagenesis methodseither introduce a selection marker in the edited locus or require atwo-step process that includes a counter-selection system. Morerecently, phage recombination proteins have been used forrecombineering, a technique that promotes homologuous recombination oflinear DNA or oligonucleotides. However, because there is no selectionof mutations, recombineering efficiency can be relatively low (0.1-10%for point mutations down to 10⁻⁵-10⁻⁶ for larger modifications), in manycases requiring the screening of a large number of colonies. Thereforenew technologies that are affordable, easy to use and efficient arestill in need for the genetic engineering of both eukaryotic andprokaryotic organisms.

Recent work on the CRISPR (clustered, regularly interspaced, shortpalindromic repeats) adaptive immune system of prokaryotes has led tothe identification of nucleases whose sequence specificity is programmedby small RNAs. CRISPR loci are composed of a series of repeats separatedby ‘spacer’ sequences that match the genomes of bacteriophages and othermobile genetic elements. The repeat-spacer array is transcribed as along precursor and processed within repeat sequences to generate smallcrRNA that specify the target sequences (also known as protospacers)cleaved by CRISPR systems. Essential for cleavage is the presence of asequence motif immediately downstream of the target region, known as theprotospacer-adjacent motif (PAM). CRISPR-associated (cas) genes usuallyflank the repeat-spacer array and encode the enzymatic machineryresponsible for crRNA biogenesis and targeting. Cas9 is a dsDNAendonuclease that uses a crRNA guide to specify the site of cleavage.Loading of the crRNA guide onto Cas9 occurs during the processing of thecrRNA precursor and requires a small RNA antisense to the precursor, thetracrRNA, and RNAse Ill. In contrast to genome editing with ZFNs orTALENs, changing Cas9 target specificity does not require proteinengineering but only the design of the short crRNA guide.

Applicants recently showed in S. pneumoniae that the introduction of aCRISPR system targeting a chromosomal locus leads to the killing of thetransformed cells. It was observed that occasional survivors containedmutations in the target region, suggesting that Cas9 dsDNA endonucleaseactivity against endogenous targets could be used for genome editing.Applicants showed that marker-less mutations can be introduced throughthe transformation of a template DNA fragment that will recombine in thegenome and eliminate Cas9 target recognition. Directing the specificityof Cas9 with several different crRNAs allows for the introduction ofmultiple mutations at the same time. Applicants also characterized indetail the sequence requirements for Cas9 targeting and show that theapproach can be combined with recombineering for genome editing in E.coli.

RESULTS: Genome Editing by Cas9 Cleavage of a Chromosomal Target

S. pneumoniae strain crR6 contains a Cas9-based CRISPR system thatcleaves a target sequence present in the bacteriophage φ8232.5. Thistarget was integrated into the srtA chromosomal locus of a second strainR6^(8232.5). An altered target sequence containing a mutation in the PAMregion was integrated into the srtA locus of a third strain R6^(370.1),rendering this strain ‘immune’ to CRISPR cleavage (FIG. 28a ).Applicants transformed R6^(8232.5) and R6^(370.1) cells with genomic DNAfrom crR6 cells, expecting that successful transformation of R6^(8232.5)cells should lead to cleavage of the target locus and cell death.Contrary to this expectation, Applicants isolated R6^(8232.5)transformants, albeit with approximately 10-fold less efficiency thanR6^(370.1) transformants (FIG. 28b ). Genetic analysis of eightR6^(8232.5) transformants (FIG. 28) revealed that the great majority arethe product of a double recombination event that eliminates the toxicityof Cas9 targeting by replacing the φ8232.5 target with the crR6 genome'swild-type srtA locus, which does not contain the protospacer requiredfor Cas9 recognition. These results were proof that the concurrentintroduction of a CRISPR system targeting a genomic locus (the targetingconstruct) together with a template for recombination into the targetedlocus (the editing template) led to targeted genome editing (FIG. 23a ).

To create a simplified system for genome editing, Applicants modifiedthe CRISPR locus in strain crR6 by deleting cas1, cas2 and csn2, geneswhich have been shown to be dispensable for CRISPR targeting, yieldingstrain crR6M (FIG. 28a ). This strain retained the same properties ofcrR6 (FIG. 28b ). To increase the efficiency of Cas9-based editing anddemonstrate that a template DNA of choice can be used to control themutation introduced, Applicants co-transformed R6^(8232.5) cells withPCR products of the wild-type srtA gene or the mutant R6^(370.1) target,either of which should be resistant to cleavage by Cas9. This resultedin a 5- to 10-fold increase of the frequency of transformation comparedwith genomic crR6 DNA alone (FIG. 23b ). The efficiency of editing wasalso substantially increased, with 8/8 transformants tested containing awild-type srtA copy and 7/8 containing the PAM mutation present in theR6^(370.1) target (FIG. 23b and FIG. 29a ). Taken together, theseresults showed the potential of genome editing assisted by Cas9.

Analysis of Cas9 Target Requirements: To introduce specific changes inthe genome, one must use an editing template carrying mutations thatabolish Cas9-mediated cleavage, thereby preventing cell death. This iseasy to achieve when the deletion of the target or its replacement byanother sequence (gene insertion) is sought. When the goal is to producegene fusions or to generate single-nucleotide mutations, the abolishmentof Cas9 nuclease activity will only be possible by introducing mutationsin the editing template that alter either the PAM or the protospacersequences. To determine the constraints of CRISPR-mediated editing,Applicants performed an exhaustive analysis of PAM and protospacermutations that abrogate CRISPR targeting.

Previous studies proposed that S. pyogenes Cas9 requires an NGG PAMimmediately downstream of the protospacer. However, because only a verylimited number of PAM-inactivating mutations have been described so far,Applicants conducted a systematic analysis to find all 5-nucleotidesequences following the protospacer that eliminate CRISPR cleavage.Applicants used randomized oligonucleotides to generate all possible1.024 PAM sequences in a heterogeneous PCR product that was transformedinto crR6 or R6 cells. Constructs carrying functional PAMs were expectedto be recognized and destroyed by Cas9 in crR6 but not R6 cells (FIG.24a ). More than 2×10⁵ colonies were pooled together to extract DNA foruse as template for the co-amplification of all targets. PCR productswere deep sequenced and found to contain all 1,024 sequences, withcoverage ranging from 5 to 42,472 reads (See section “Analysis of deepsequencing data”). The functionality of each PAM was estimated by therelative proportion of its reads in the crR6 sample over the R6 sample.Analysis of the first three bases of the PAM, averaging over the twolast bases, clearly showed that the NGG pattern was under-represented incrR6 transformants (FIG. 24b ). Furthermore, the next two bases had nodetectable effect on the NGG PAM (See section “Analysis of deepsequencing data”), demonstrating that the NGGNN sequence was sufficientto license Cas9 activity. Partial targeting was observed for NAG PAMsequences (FIG. 24b ). Also the NNGGN pattern partially inactivatedCRISPR targeting (Table G), indicating that the NGG motif can still berecognized by Cas9 with reduced efficiency when shifted by 1 bp. Thesedata shed light onto the molecular mechanism of Cas9 target recognition,and they revealed that NGG (or CCN on the complementary strand)sequences are sufficient for Cas9 targeting and that NGG to NAG or NNGGNmutations in the editing template should be avoided. Owing to the highfrequency of these tri-nucleotide sequences (once every 8 bp), thismeans that almost any position of the genome can be edited. Indeed,Applicants tested ten randomly chosen targets carrying various PAMs andall were found to be functional (FIG. 30).

Another way to disrupt Cas9-mediated cleavage is to introduce mutationsin the protospacer region of the editing template. It is known thatpoint mutations within the ‘seed sequence’ (the 8 to 10 protospacernucleotides immediately adjacent to the PAM) can abolish cleavage byCRISPR nucleases. However, the exact length of this region is not known,and it is unclear whether mutations to any nucleotide in the seed candisrupt Cas9 target recognition. Applicants followed the same deepsequencing approach described above to randomize the entire protospacersequence involved in base pair contacts with the crRNA and to determineall sequences that disrupt targeting. Each position of the 20 matchingnucleotides (14) in the spc1 target present in R6^(8232.5) cells (FIG.23a ) was randomized and transformed into crR6 and R6 cells (FIG. 24a ).Consistent with the presence of a seed sequence, only mutations in the12 nucleotides immediately upstream of the PAM abrogated cleavage byCas9 (FIG. 24c ). However, different mutations displayed markedlydifferent effects. The distal (from the PAM) positions of the seed (12to 7) tolerated most mutations and only one particular base substitutionabrogated targeting. In contrast, mutations to any nucleotide in theproximal positions (6 to 1, except 3) eliminated Cas9 activity, althoughat different levels for each particular substitution. At position 3,only two substitutions affected CRISPR activity and with differentstrength. Applicants concluded that, although seed sequence mutationscan prevent CRISPR targeting, there are restrictions regarding thenucleotide changes that can be made in each position of the seed.Moreover, these restrictions can most likely vary for different spacersequences. Therefore Applicants believe that mutations in the PAMsequence, if possible, should be the preferred editing strategy.Alternatively, multiple mutations in the seed sequence may be introducedto prevent Cas9 nuclease activity.

Cas9-Mediated Genome Editing in S. pneumonia: To develop a rapid andefficient method for targeted genome editing, Applicants engineeredstrain crR6Rk, a strain in which spacers can be easily introduced by PCR(FIG. 33). Applicants decided to edit the β-galactosidase (bgaA) gene ofS. pneumoniae, whose activity can be easily measured. Applicantsintroduced alanine substitutions of amino acids in the active site ofthis enzyme: R481A (R→A) and N563A,E564A (NE→AA) mutations. Toillustrate different editing strategies, Applicants designed mutationsof both the PAM sequence and the protospacer seed. In both cases thesame targeting construct with a crRNA complementary to a region of theβ-galactosidase gene that is adjacent to a TGG PAM sequence (CCA in thecomplementary strand, FIG. 26) was used. The R→A editing templatecreated a three-nucleotide mismatch on the protospacer seed sequence(CGT to GCA, also introducing a BtgZI restriction site). In the NE→AAediting template Applicants simultaneously introduced a synonymousmutation that created an inactive PAM (TGG to TTG) along with mutationsthat are 218 nt downstream of the protospacer region (AAT GAA to GCTGCA, also generating a TseI restriction site). This last editingstrategy demonstrated the possibility of using a remote PAM to makemutations in places where a proper target may be hard to choose. Forexample, although the S. pneumoniae R6 genome, which has a 39.7% GCcontent, contains on average one PAM motif every 12 bp, some PAM motifsare separated by up to 194 bp (FIG. 33). In addition Applicants designeda ΔbgaA in-frame deletion of 6,664 bp. In all three cases,co-transformation of the targeting and editing templates produced10-times more kanamycin-resistant cells than co-transformation with acontrol editing template containing wild-type bgaA sequences (FIG. 25b). Applicants genotyped 24 transformants (8 for each editing experiment)and found that all but one incorporated the desired change (FIG. 25c ).DNA sequencing also confirmed not only the presence of the introducedmutations but also the absence of secondary mutations in the targetregion (FIG. 29b,c ). Finally, Applicants measured γ-galactosidaseactivity to confirm that all edited cells displayed the expectedphenotype (FIG. 25d ).

Cas9-mediated editing can also be used to generate multiple mutationsfor the study of biological pathways. Applicants decided to illustratethis for the sortase-dependent pathway that anchors surface proteins tothe envelope of Gram-positive bacteria. Applicants introduced a sortasedeletion by co-transformation of a chloramphenicol-resistant targetingconstruct and a ΔsrtA editing template (FIG. 33a,b ), followed by aΔbgaA deletion using a kanamycin-resistant targeting construct thatreplaced the previous one. In S. pneumoniae, β-galactosidase iscovalently linked to the cell wall by sortase. Therefore, deletion ofsrtA results in the release of the surface protein into the supernatant,whereas the double deletion has no detectable β-galactosidase activity(FIG. 34c ). Such a sequential selection can be iterated as many timesas required to generate multiple mutations.

These two mutations may also be introduced at the same time. Applicantsdesigned a targeting construct containing two spacers, one matching srtAand the other matching bgaA, and co-transformed it with both editingtemplates at the same time (FIG. 25e ). Genetic analysis oftransformants showed that editing occurred in 6/8 cases (FIG. 25f ).Notably, the remaining two clones each contained either a ΔsrtA or aΔbgaA deletion, suggesting the possibility of performing combinatorialmutagenesis using Cas9. Finally, to eliminate the CRISPR sequences,Applicants introduced a plasmid containing the bgaA target and aspectinomycin resistance gene along with genomic DNA from the wild-typestrain R6. Spectinomycin-resistant transformants that retain the plasmideliminated the CRISPR sequences (FIG. 34a,d ).

Mechanism and Efficiency of Editing: To understand the mechanismsunderlying genome editing with Cas9, Applicants designed an experimentin which the editing efficiency was measured independently of Cas9cleavage. Applicants integrated the ermAM erythromycin resistance genein the srtA locus, and introduced a premature stop codon usingCas9-mediated editing (FIG. 33). The resulting strain (JEN53) containsan ermAM(stop) allele and is sensitive to erythromycin. This strain maybe used to assess the efficiency at which the ermAM gene is repaired bymeasuring the fraction of cells that restore antibiotic resistance withor without the use of Cas9 cleavage. JEN53 was transformed with anediting template that restores the wild-type allele, together witheither a kanamycin-resistant CRISPR construct targeting the ermAM(stop)allele (CRISPR::ermAM(stop)) or a control construct without a spacer(CRISPR::Ø) (FIG. 26a,b ). In the absence of kanamycin selection, thefraction of edited colonies was on the order of 10⁻²(erythromycin-resistant cfu/total cfu) (FIG. 26c ), representing thebaseline frequency of recombination without Cas9-mediated selectionagainst unedited cells. However, if kanamycin selection was applied andthe control CRISPR construct was co-transformed, the fraction of editedcolonies increased to about 10⁻¹ (kanamycin- and erythromycin-resistantcfu/kanamycin-resistant cfu) (FIG. 26c ). This result shows thatselection for the recombination of the CRISPR locus co-selected forrecombination in the ermAM locus independently of Cas9 cleavage of thegenome, suggesting that a subpopulation of cells is more prone totransformation and/or recombination. Transformation of theCRISPR::ermAM(stop) construct followed by kanamycin selection resultedin an increase of the fraction of erythromycin-resistant, edited cellsto 99% (FIG. 26c ). To determine if this increase is caused by thekilling of non-edited cells, Applicants compared the kanamycin-resistantcolony forming units (cfu) obtained after co-transformation of JEN53cells with the CRISPR::ermAM(stop) or CRISPR::Ø constructs.

Applicants counted 5.3 times less kanamycin-resistant colonies aftertransformation of the ermAM(stop) construct (2.5×10⁴/4.7−10³, FIG. 35a), a result that suggests that indeed targeting of a chromosomal locusby Cas9 leads to the killing of non-edited cells. Finally, because theintroduction of dsDNA breaks in the bacterial chromosome is known totrigger repair mechanisms that increase the rate of recombination of thedamaged DNA, Applicants investigated whether cleavage by Cas9 inducesrecombination of the editing template. Applicants counted 2.2 times morecolonies after co-transformation with the CRISPR::erm(stop) constructthan with the CRISPR::Ø construct (FIG. 26d ), indicating that there wasa modest induction of recombination. Taken together, these resultsshowed that co-selection of transformable cells, induction ofrecombination by Cas9-mediated cleavage and selection against non-editedcells, each contributed to the high efficiency of genome editing in S.pneumoniae.

As cleavage of the genome by Cas9 should kill non-edited cells, onewould not expect to recover any cells that received the kanamycinresistance-containing Cas9 cassette but not the editing template.However, in the absence of the editing template Applicants recoveredmany kanamycin-resistant colonies after transformation of theCRISPR::ermAM(stop) construct (FIG. 35a ). These cells that ‘escape’CRISPR-induced death produced a background that determined a limit ofthe method. This background frequency may be calculated as the ratio ofCRISPR::ermA M(stop)/CRISPR::Ø cfu, 2.6×10⁻³ (7.1×10¹/2.7×10⁴) in thisexperiment, meaning that if the recombination frequency of the editingtemplate is less than this value, CRISPR selection may not efficientlyrecover the desired mutants above the background. To understand theorigin of these cells, Applicants genotyped 8 background colonies andfound that 7 contained deletions of the targeting spacer (FIG. 35b ) andone harbored a presumably inactivating mutation in Cas9 (FIG. 35c ).

Genome Editing with Cas9 in E. coli: The activation of Cas9 targetingthrough the chromosomal integration of a CRISPR-Cas system is onlypossible in organisms that are highly recombinogenic. To develop a moregeneral method that is applicable to other microbes, Applicants decidedto perform genome editing in E. coli using a plasmid-based CRISPR-Cassystem. Two plasmids were constructed: a pCas9 plasmid carrying thetracrRNA, Cas9 and a chloramphenicol resistance cassette (FIG. 36), anda pCRISPR kanamycin-resistant plasmid carrying the array of CRISPRspacers. To measure the efficiency of editing independently of CRISPRselection, Applicants sought to introduce an A to C transversion in therpsL gene that confers streptomycin resistance. Applicants constructed apCRISPR::rpsL plasmid harboring a spacer that would guide Cas9 cleavageof the wild-type, but not the mutant rpsL allele (FIG. 27b ). The pCas9plasmid was first introduced into E. coli MG1655 and the resultingstrain was co-transformed with the pCRISPR::rpsL plasmid and W542, anediting oligonucleotide containing the A to C mutation.streptomycin-resistant colonies after transformation of thepCRISPR::rpsL plasmid were only recovered, suggesting that Cas9 cleavageinduces recombination of the oligonucleotide (FIG. 37). However, thenumber of streptomycin-resistant colonies was two orders of magnitudelower than the number of kanamycin-resistant colonies, which arepresumably cells that escape cleavage by Cas9. Therefore, in theseconditions, cleavage by Cas9 facilitated the introduction of themutation, but with an efficiency that was not enough to select themutant cells above the background of ‘escapers’.

To improve the efficiency of genome editing in E. coli, Applicantsapplied their CRISPR system with recombineering, using Cas9-induced celldeath to select for the desired mutations. The pCas9 plasmid wasintroduced into the recombineering strain HME63 (31), which contains theGam, Exo and Beta functions of the □-red phage. The resulting strain wasco-transformed with the pCRISPR::rpsL plasmid (or a pCRISPR::Ø control)and the W542 oligonucleotide (FIG. 27a ). The recombineering efficiencywas 5.3 10−5, calculated as the fraction of total cells that becomestreptomycin-resistant when the control plasmid was used (FIG. 27c ). Incontrast, transformation with the pCRISPR::rpsL plasmid increased thepercentage of mutant cells to 65±14% (FIGS. 27c and 29f ). Applicantsobserved that the number of cfu was reduced by about three orders ofmagnitude after transformation of the pCRISPR::rpsL plasmid than thecontrol plasmid (4.8×10⁵/5.3×10², FIG. 38a ), suggesting that selectionresults from CRISPR-induced death of non-edited cells. To measure therate at which Cas9 cleavage was inactivated, an important parameter ofApplicants' method, Applicants transformed cells with eitherpCRISPR::rpsL or the control plasmid without the W542 editingoligonucleotide (FIG. 38a ). This background of CRISPR ‘escapers’,measured as the ratio of pCRISPR::rpsL/pCRISPR::Ø cfu, was 2.5×10⁻⁴(1.2×10⁵/4.8×10⁵). Genotyping eight of these escapers revealed that inall cases there was a deletion of the targeting spacer (FIG. 38b ). Thisbackground was higher than the recombineering efficiency of the rpsLmutation, 5.3×10⁻⁵, which suggested that to obtain 65% of edited cells,Cas9 cleavage must induce oligonucleotide recombination. To confirmthis, Applicants compared the number of kanamycin- andstreptomycin-resistant cfu after transformation of pCRISPR::rpsL orpCRISPR::Ø (FIG. 27d ). As in the case for S. pneumoniae, Applicantsobserved a modest induction of recombination, about 6.7 fold(2.0×10⁻⁴/3.0×10⁻⁵). Taken together, these results indicated that theCRISPR system provided a method for selecting mutations introduced byrecombineering.

Applicants showed that CRISPR-Cas systems may be used for targetedgenome editing in bacteria by the co-introduction of a targetingconstruct that killed wild-type cells and an editing template that botheliminated CRISPR cleavage and introduced the desired mutations.Different types of mutations (insertions, deletions or scar-lesssingle-nucleotide substitutions) may be generated. Multiple mutationsmay be introduced at the same time. The specificity and versatility ofediting using the CRISPR system relied on several unique properties ofthe Cas9 endonuclease: (i) its target specificity may be programmed witha small RNA, without the need for enzyme engineering. (ii) targetspecificity was very high, determined by a 20 bp RNA-DNA interactionwith low probability of non-target recognition, (iii) almost anysequence may be targeted, the only requirement being the presence of anadjacent NGG sequence, (iv) almost any mutation in the NGG sequence, aswell as mutations in the seed sequence of the protospacer, eliminatestargeting.

Applicants showed that genome engineering using the CRISPR system workednot only in highly recombinogenic bacteria such as S. pneumoniae, butalso in E. coli. Results in E. coli suggested that the method may beapplicable to other microorganisms for which plasmids may be introduced.In E. coli, the approach complements recombineering of mutagenicoligonucleotides. To use this methodology in microbes whererecombineering is not a possible, the host homologous recombinationmachinery may be used by providing the editing template on a plasmid. Inaddition, because accumulated evidence indicates that CRISPR-mediatedcleavage of the chromosome leads to cell death in many bacteria andarchaea, it is possible to envision the use of endogenous CRISPR-Cassystems for editing purposes.

In both S. pneumoniae and E. coli, Applicants observed that althoughediting was facilitated by a co-selection of transformable cells and asmall induction of recombination at the target site by Cas9 cleavage,the mechanism that contributed the most to editing was the selectionagainst non-edited cells. Therefore the major limitation of the methodwas the presence of a background of cells that escape CRISPR-inducedcell death and lack the desired mutation. Applicants showed that these‘escapers’ arose primarily through the deletion of the targeting spacer,presumably after the recombination of the repeat sequences that flankthe targeting spacer. Future improvements may focus on the engineeringof flanking sequences that can still support the biogenesis offunctional crRNAs but that are sufficiently different from one anotherto eliminate recombination. Alternatively, the direct transformation ofchimeric crRNAs may be explored. In the particular case of E. coli, theconstruction of the CRISPR-Cas system was not possible if this organismwas also used as a cloning host. Applicants solved this issue by placingCas9 and the tracrRNA on a different plasmid than the CRISPR array. Theengineering of an inducible system may also circumvent this limitation.

Although new DNA synthesis technologies provide the ability tocost-effectively create any sequence with a high throughput, it remainsa challenge to integrate synthetic DNA in living cells to createfunctional genomes. Recently, the co-selection MAGE strategy was shownto improve the mutation efficiency of recombineering by selecting asubpopulation of cells that has an increased probability to achieverecombination at or around a given locus. In this method, theintroduction of selectable mutations is used to increase the chances ofgenerating nearby non-selectable mutations. As opposed to the indirectselection provided by this strategy, the use of the CRISPR system makesit possible to directly select for the desired mutation and to recoverit with a high efficiency. These technologies add to the toolbox ofgenetic engineers, and together with DNA synthesis, they maysubstantially advance both the ability to decipher gene function and tomanipulate organisms for biotechnological purposes. Two other studiesalso relate to CRISPR-assisted engineering of mammalian genomes. It isexpected that these crRNA-directed genome editing technologies may bebroadly useful in the basic and medical sciences.

Strains and Culture Conditions. S. pneumoniae strain R6 was provided byDr. Alexander Tomasz. Strain crR6 was generated in a previous study.Liquid cultures of S. pneumoniae were grown in THYE medium (30 g/lTodd-Hewitt agar, 5 g/l yeast extract). Cells were plated on tryptic soyagar (TSA) supplemented with 5% defibrinated sheep blood. Whenappropriate, antibiotics were added as followings: kanamycin (400μg/ml), chloramphenicol (5 μg/ml), erythromycin (1 μg/ml) streptomycin(100 μg/ml) or spectinomycin (100 μg/ml). Measurements ofβ-galactosidase activity were made using the Miller assay as previouslydescribed.

E. coli strains MG1655 and HME63 (derived from MG1655, Δ(argF-lac) U169λ cI857 Δcro-bioA galK tyr 145 UAG mutS<>amp) (31) were provided by JeffRoberts and Donald Court, respectively. Liquid cultures of E. coli weregrown in LB medium (Difco). When appropriate, antibiotics were added asfollowings: chloramphenicol (25 μg/ml), kanamycin (25 μg/ml) andstreptomycin (50 μg/ml).

S. pneumoniae Transformation. Competent cells were prepared as describedpreviously (23). For all genome editing transformations, cells weregently thawed on ice and resuspended in 10 volumes of M2 mediumsupplemented with 100 ng/ml of competence-stimulating peptide CSP1(40),and followed by addition of editing constructs (editing constructs wereadded to cells at a final concentration between 0.7 ng/μl to 2.5 μg/ul).Cells were incubated 20 min at 37° C. before the addition of 2 μl oftargeting constructs and then incubated 40 min at 37° C. Serialdilutions of cells were plated on the appropriate medium to determinethe colony forming units (cfu) count.

E. coli Lambda-red Recombineering. Strain HME63 was used for allrecombineering experiments. Recombineering cells were prepared andhandled according to a previously published protocol (6). Briefly, a 2ml overnight culture (LB medium) inoculated from a single colonyobtained from a plate was grown at 30° C. The overnight culture wasdiluted 100-fold and grown at 30° C. with shaking (200 rpm) until theOD₆₀₀ is from 0.4-0.5 (approximately 3 hrs). For Lambda-red induction,the culture was transferred to a 42° C. water bath to shake at 200 rpmfor 15 min. Immediately after induction, the culture was swirled in anice-water slurry and chilled on ice for 5-10 min. Cells were then washedand aliquoted according to the protocol. For electro-transformation, 50μl of cells were mixed with 1 mM of salt-free oligos (IDT) or 100-150 ngof plasmid DNA (prepared by QIAprep Spin Miniprep Kit, Qiagen). Cellswere electroporated using 1 mm Gene Pulser cuvette (Bio-rad) at 1.8 kVand were immediately resuspended in 1 ml of room temperature LB medium.Cells were recovered at 30° C. for 1-2 hrs before being plated on LBagar with appropriate antibiotic resistance and incubated at 32° C.overnight.

Preparation of S. pneumoniae Genomic DNA. For transformation purposes,S. pneumoniae genomic DNA was extracted using the Wizard Genomic DNAPurification Kit, following instructions provided by the manufacturer(Promega). For genotyping purposes, 700 ul of overnight S. pneumoniaecultures were pelleted, resuspended in 60 ul of lysozyme solution (2mg/ml) and incubated 30 min at 37° C. The genomic DNA was extractedusing QIAprep Spin Miniprep Kit (Qiagen).

Strain Construction. All primers used in this study are provided inTable G. To generate S. pneumoniae crR6M, an intermediate strain,LAM226, was made. In this strain the aphA4-3 gene (providing kanamycinresistance) adjacent to the CRISPR array of S. pneumoniae crR6 strainwas replaced by a cat gene (providing chloramphenicol resistance).Briefly, crR6 genomic DNA was amplified using primers L448/L444 andL447/L481, respectively. The cat gene was amplified from plasmid pC194using primers L445/L446. Each PCR product was gel-purified and all threewere fused by SOEing PCR with primers L448/L481. The resulting PCRproduct was transformed into competent S. pneumoniae crR6 cells andchloramphenicol-resistant transformants were selected. To generate S.pneumoniae crR6M, S. pneumoniae crR6 genomic DNA was amplified by PCRusing primers L409/L488 and L448/L481, respectively. Each PCR productwas gel-purified and they were fused by SOEing PCR with primersL409/L481. The resulting PCR product was transformed into competent S.pneumoniae LAM226 cells and kanamycin-resistant transformants wereselected.

To generate S. pneumoniae crR6Rc, S. pneumoniae crR6M genomic DNA wasamplified by PCR using primers L430/W286, and S. pneumoniae LAM226genomic DNA was amplified by PCR using primers W288/L481. Each PCRproduct was gel-purified and they were fused by SOEing PCR with primersL430/L481. The resulting PCR product was transformed into competent S.pneumoniae crR6M cells and chloramphenicol-resistant transformants wereselected.

To generate S. pneumoniae crR6Rk, S. pneumoniae crR6M genomic DNA wasamplified by PCR using primers L430/W286 and W287/L481, respectively.Each PCR product was gel-purified and they were fused by SOEing PCR withprimers L430/L481. The resulting PCR product was transformed intocompetent S. pneumoniae crR6Rc cells and kanamycin-resistanttransformants were selected.

To generate JEN37, S. pneumoniae crR6Rk genomic DNA was amplified by PCRusing primers L430/W356 and W357/L481, respectively. Each PCR productwas gel-purified and they were fused by SOEing PCR with primersL430/L481. The resulting PCR product was transformed into competent S.pneumoniae crR6Rc cells and kanamycin-resistant transformants wereselected.

To generate JEN38, R6 genomic DNA was amplified using primers L422/L461and L459/L426, respectively. The ermAM gene (specifying erythromycinresistance) was amplified from plasmid pFW5 ⁴³ using primers L457/L458.Each PCR product was gel-purified and all three were fused by SOEing PCRwith primers L422/L426. The resulting PCR product was transformed intocompetent S. pneumoniae crR6Rc cells and erythromycin-resistanttransformants were selected.

S. pneumoniae JEN53 was generated in two steps. First JEN43 wasconstructed as illustrated in FIG. 33. JEN53 was generated bytransforming genomic DNA of JEN25 into competent JEN43 cells andselecting on both chloramphenicol and erythromycin.

To generate S. pneumoniae JEN62, S. pneumoniae crR6Rk genomic DNA wasamplified by PCR using primers W256/W365 and W366/L403, respectively.Each PCR product was purified and ligated by Gibson assembly. Theassembly product was transformed into competent S. pneumoniae crR6Rccells and kanamycin-resistant transformants were selected.

Plasmid Construction. pDB97 was constructed through phosphorylation andannealing of oligonucleotides B296/B297, followed by ligation inpLZ12spec digested by EcoRI/BamHI. Applicants fully sequenced pLZ12specand deposited its sequence in genebank (accession: KC 112384).

pDB98 was obtained after cloning the CRISPR leader sequence was clonedtogether with a repeat-spacer-repeat unit into pLZ12spec. This wasachieved through amplification of crR6Rc DNA with primers B298/B320 andB299/B321, followed by SOEing PCR of both products and cloning inpLZ12spec with restriction sites BamHI/EcoRI. In this way the spacersequence in pDB98 was engineered to contain two BsaI restriction sitesin opposite directions that allow for the scar-less cloning of newspacers.

pDB99 to pDB108 were constructed by annealing of oligonucleotidesB300/B301 (pDB99). B302/B303 (pDB100), B304/B305 (pDB001). B306/B307(pDB102), B308/B309 (pDB103), B310/B311 (pDB104), B312/B313 (pDB105),B314/B315 (pDB106), B315/B317 (pDB107), B318/B319 (pDB108), followed byligation in pDB98 cut by BsaI.

The pCas9 plasmid was constructed as follow. Essential CRISPR elementswere amplified from Streptococcos pyogenes SF370 genomic DNA withflanking homology arms for Gibson Assembly. The tracrRNA and Cas9 wereamplified with oligos HC008 and HC010. The leader and CRISPR sequenceswere amplified HC011/HC014 and HC015/HC009, so that two BsaI type IISsites were introduced in between two direct repeats to facilitate easyinsertion of spacers.

pCRISPR was constructed by subcloning the pCas9 CRISPR array inpZE21-MCS1 through amplification with oligos B298+B299 and restrictionwith EcoRI and BamHI. The rpsL targeting spacer was cloned by annealingof oligos B352+B353 and cloning in the BsaI cut pCRISPR givingpCRISPR::rpsL.

Generation of Targeting and Editing Constructs. Targeting constructsused for genome editing were made by Gibson assembly of Left PCRs andRight PCRs (Table G). Editing constructs were made by SOEing PCR fusingPCR products A (PCR A), PCR products B (PCR B) and PCR products C (PCRC) when applicable (Table G). The CRISPR::Ø and CRISPR::ermAM(stop)targeting constructs were generated by PCR amplification of JEN62 andcrR6 genomic DNA respectively, with oligos L409 and L481.

Generation of Targets with Randomized PAM or Protospacer Sequences. The5 nucleotides following the spacer 1 target were randomized throughamplification of R6^(8232.5) genomic DNA with primers W377/L426. ThisPCR product was then assembled with the cat gene and the srtA upstreamregion that were amplified from the same template with primersL422/W376. 80 ng of the assembled DNA was used to transform strains R6and crR6. Samples for the randomized targets were prepared using thefollowing primers: B280-B290/L426 to randomize bases 1-10 of the targetand B269-B278/L426 to randomize bases 10-20. Primers L422/B268 andL422/B279 were used to amplify the cat gene and srtA upstream region tobe assembled with the first and last 10 PCR products respectively. Theassembled constructs were pooled together and 30 ng was transformed inR6 and crR6. After transformation, cells were plated on chloramphenicolselection. For each sample more than 2×10⁵ cells were pooled together in1 ml of THYE and genomic DNA was extracted with the Promega Wizard kit.Primers B250/B251 were used to amplify the target region. PCR productswere tagged and run on one Illumina MiSeq paired-end lane using 300cycles.

Analysis of Deep Sequencing Data.

Randomized PAM: For the randomized PAM experiment 3,429,406 reads wereobtained for crR6 and 3,253,998 for R6. It is expected that only half ofthem will correspond to the PAM-target while the other half willsequence the other end of the PCR product. 1,623,008 of the crR6 readsand 1,537,131 of the R6 reads carried an error-free target sequence. Theoccurrence of each possible PAM among these reads is shown insupplementary file. To estimate the functionality of a PAM, its relativeproportion in the crR6 sample over the R6 sample was computed and isdenoted r_(ijklm) where Ij,k,l,m are one of the 4 possible bases. Thefollowing statistical model was constructed:log(r _(ijklm))=μ+b2_(i) +b3_(j) +b4_(k) +b2b3_(i,j)+b3b4_(j,k)+ε_(ijklm),

where ε is the residual error, b2 is the effect of the 2^(nd) base ofthe PAM, b3 of the third, b4 of the fourth, b2b3 is the interactionbetween the second and third bases, b3b4 between the third and fourthbases. An analysis of variance was performed:

Anova table Df Sum Sq Mean Sq F value Pr(>F) b3 3 151.693 50.564601.8450 <2.2e−16 *** b2 3 90.521 30.174 359.1454 <2.2e−16 *** b4 31.881 0.627 7.4623 6.070e−05 ***  b3:b2 9 228.940 25.438 302.7738<2.2e−16 *** b3:b4 9 3.010 0.334 3.9809 5.227e−05 ***  Residuals 99683.680 0.084

When added to this model, b1 or b5 do not appear to be significant andother interactions than the ones included can also be discarded. Themodel choice was made through successive comparisons of more or lesscomplete models using the anova method in R. Tukey's honest significancetest was used to determine if pairwise differences between effects aresignificant.

NGGNN patterns are significantly different from all other patterns andcarry the strongest effect (see table below).

In order to show that positions 1, 4 or 5 do not affect the NGGNNpattern Applicants looked at theses sequences only. Their effect appearsto be normally distributed (see QQ plot in FIG. 71), and modelcomparisons using the anova method in R shows that the null model is thebest one, i.e. there is no significant role of b1, b4 and b5.

Model Comparison Using the Anova Method in R for the NGGNN Sequences

Model 1: ratio.log~1 Model 2: ratio.log~b1 + b4 + b5 Res. Df RSS Df Sumof Sq F Pr (>F) 1 63 14.579 2 54 11.295 9 3.2836 1.7443 0.1013

Partial Interference of NAGNN and NNGGN Patterns

NAGNN patterns are significantly different from all other patterns butcarry a much smaller effect than NGGNN (see Tukey's honest significancetest below).

Finally, NTGGN and NCGGN patterns are similar and show significantlymore CRISPR interference than NTGHN and NCGHN patterns (where H is A, Tor C), as shown by a bonferroni adjusted pairwise student-test.

Pairwise Comparisons of the Effect of b4 on NYGNN Sequences Using tTests with Pooled SD

Data: b4 A C G C 1.00 — — G 9.2e−05 2.4e−06 — T 0.31 1.00 1.2e−08

Taken together, these results allow concluding that NNGGN patterns ingeneral produce either a complete interference in the case of NGGGN, ora partial interference in the case of NAGGN, NTGGN or NCGGN.

Tukey multiple comparisons of means: 95% family-wise confidence level

diff lwr upr p adj $b2:b3 G:G-A:A −2.76475 −2.94075 −2.58875 <1E−07G:G-C:A −2.79911 −2.97511 −2.62311 <1E−07 G:G-T:A −2.7809 −2.9569−2.6049 <1E−07 G:G-A:C −2.81643 −2.99244 −2.64043 <1E−07 G:G-C:C−2.77903 −2.95504 −2.60303 <1E−07 G:G-G:C −2.64867 −2.82468 −2.47267<1E−07 G:G-T:C −2.79718 −2.97319 −2.62118 <1E−07 G:G-A:G −2.67068−2.84668 −2.49468 <1E−07 G:G-C:G −2.73525 −2.91125 −2.55925 <1E−07G:G-T:G −2.7976 −2.62159 −2.9736 <1E−07 G:G-A:T −2.76727 −2.59127−2.94328 <1E−07 G:G-C:T −2.84114 −2.66513 −3.01714 <1E−07 G:G-G:T−2.76409 −2.58809 −2.94009 <1E−07 G:G-T:T −2.76781 −2.59181 −2.94381<1E−07 G:G-G:A −2.13964 −2.31565 −1.96364 <1E−07 G:A-A:A −0.62511−0.80111 −0.4491 <1E−07 G:A-C:A −0.65947 −0.83547 −0.48346 <1E−07G:A-T:A −0.64126 −0.46525 −0.81726 <1E−07 G:A-A:C −0.67679 −0.50078−0.85279 <1E−07 G:A-C:C −0.63939 −0.46339 −0.81539 <1E−07 G:A-G:C−0.50903 −0.33303 −0.68503 <1E−07 G:A-T:C −0.65754 −0.48154 −0.83354<1E−07 G:A-A:G −0.53104 −0.35503 −0.70704 <1E−07 G:A-C:G −0.59561−0.4196 −0.77161 <1E−07 G:A-T:G −0.65795 −0.48195 −0.83396 <1E−07G:A-A:T −0.62763 −0.45163 −0.80363 <1E−07 G:A-C:T −0.70149 −0.52549−0.8775 <1E−07 G:A-G:T −0.62445 −0.44844 −0.80045 <1E−07 G:A-T:T−0.62817 −0.45216 −0.80417 <1E−07 $b3:b4 G:G-G:A −0.33532 −0.51133−0.15932 <1E−07 G:G-G:C −0.18118 −0.35719 −0.00518 0.036087 G:G-G:T−0.31626 −0.14026 −0.49226 <1E−07

Randomized Target

For the randomized target experiment 540,726 reads were obtained forcrR6 and 753,570 for R6. As before, only half of the reads are expectedto sequence the interesting end of the PCR product. After filtering forreads that carry a target that is error-free or with a single pointmutation, 217,656 and 353,141 reads remained for crR6 and R6respectively. The relative proportion of each mutant in the crR6 sampleover the R6 sample was computed (FIG. 24c ). All mutations outside ofthe seed sequence (13-20 bases away from the PAM) show fullinterference. Those sequences were used as a reference to determine ifother mutations inside the seed sequence can be said to significantlydisrupt interference. A normal distribution was fitted to thesessequences using the fitdistr function of the MASS R package. The 0.99quantile of the fitted distribution is shown as a dotted line in FIG.24c . FIG. 72 shows a histogram of the data density with fitted normaldistribution (black line) and 0.99 quantile (dotted line).

TABLE F Relative abundance of PAM sequences in the crR6/R6 samplesaveraged over bases 1 and 5. 3rd position A C G T 2nd A AAA 1.04 ACA1.12 AGA 0.73 ATA 1.10 A 4th position AAC 1.07 ACC 1.04 AGC 0.64 ATC0.97 C position AAG 1.00 ACG 1.09 AGG 0.61 ATG 1.07 G AAT 0.98 ACT 1.02AGT 0.65 ATT 1.01 T C CAA 1.05 CCA 1.05 CGA 0.99 CTA 1.07 A CAC 1.04 CCC1.02 CGC 1.08 CTC 1.04 C CAG 1.08 CCG 1.08 CGG 0.81 CTG 1.05 G CAT 1.13CCT 1.05 CGT 1.07 CTT 1.08 T G GAA 0.97 GCA 1.05 GGA 0.08 GTA 0.99 A GAC0.92 GCC 1.00 GGC 0.05 GTC 1.15 C GAG 0.96 GCG 0.98 GGG 0.07 GTG 0.98 GGAT 0.98 GCT 0.99 GGT 0.06 GTT 1.05 T T TAA 1.08 TCA 1.16 TGA 1.05 TTA1.14 A TAC 1.00 TCC 1.08 TGC 1.08 TTC 1.05 C TAG 1.02 TCG 1.11 TGG 0.77TTG 1.01 G TAT 1.01 TCT 1.12 TGT 1.21 TTT 1.02 T

TABLE GPrimers used in this study (SEQ ID NOS 68-183, respectively, in order ofappearance). Primer Sequence 5′-3′ B217TCCTAGCAGGATTTCTGATATTACTGTCACGTTTTAGAGCTATGCTGTTTTGA B218GTGACAGTAATATCAGAAATCCTGCTAGGAGTTTTGGGACCATTCAAACAGC B229GGGTTTCAAGTCTTTGTAGCAAGAG B230 GCCAATGAACGGGAACCCTTGGTC B250NNNNGACGAGGCAATGGCTGAAATC B251 NNNNTTATTTGGCTCATATTTGCTG B255CTTTACACCAATCGCTGCAACAGAC B256CAAAATTTCTAGTGTTCTTTGCCTTTCCCCATAAAACCCTCCTTA B257AGGGTTTTATGGGGAAAGGCAAAGAAGACTAGAAATTTTGATACC B258CTTACGGTGCATAAAGTCAATTTCC B269 TGGCTCGATTTCAGCCATTGC B270CTTTGACGAGGCAATGGCTGAAATCGAGCCAANAAAGCGCAAG B271CTTTGACGAGGCAATGGCTGAAATCGAGCCAAANAAGCGCAAG B272CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAANAGCGCAAG B273CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAANGCGCAAG B274CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAANCGCAAG B275CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAAGNGCAAG B276CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAAGCNCAAGAAG B277CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAAGCGNAAGAAG B278CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAAGCGCNAGAAG B279GCGCTTTTTTGGCTCGATTTCAG B280 CAATGGCTGAAATCGAGCCAAAAAAGCGCANGAAGAAATCB281 CAATGGCTGAAATCGAGCCAAAAAAGCGCAANAAGAAATC B282CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGNAGAAATC B283CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGANGAAATC B284CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAANAAATC B285CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGNAATCAACC B286CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGANATCAACC B287CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGAANTCAACC B288CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGAAANCAACC B289CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGAAATNAACCAGC B290CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGAAATCNACCAGC B296gatccTCCATCCGTACAACCCACAACCCTGg B297 aattcCAGGGTTGTGGGTTGTACGGATGGAgB298 CATGGATCCTATTTCTTAATAACTAAAAATATGG B299CATGAATTCAACTCAACAAGTCTCAGTGTGCTG B300AAACATTTTTTCTCCATTTAGGAAAAAGGATGCTG B301AAAACAGCATCCTTTTTCCTAAATGGAGAAAAAAT B302AAACCTTAAATCACTCACAAATAGCAGCAAAATTG B303AAAACAATTTTGCTGCTATTTGTGACTGATTTAAG B304AAACTTTTCATCATACGACCAATCTGCTTTATTTG B305AAAACAAATAAAGCAGATTGGTCGTATGATGAAAA B306AAACTCGTCCAGAAGTTATCGTAAAAGAAATCGAG B307AAAACTCGATTTCTTTTACGATAACTTCTGGACGA B308AAACAATCTCTCCAAGGTTTCCTTAAAAATCTCTG B309AAAACAGAGATTTTTAAGGAAACCTTGGAGAGATT B310AAACGCCATCGTCAGGAAGAAGCTATGCTTGAGTG B311AAAACACTCAAGCATAGCTTCTTCCTGACGATGGC B312AAACATCTCTATACTTATTGAAATTTCTTTGTATG B313AAAACATACAAAGAAATTTCAATAAGTATAGAGAT B314AAACTAGCTGTGATAGTCCGCAAAACCAGCCTTCG B315AAAACGAAGGCTGGTTTTGCGGACTATCACAGCTA B316AAACATCGGAAGGTCGAGCAAGTAATTATCTTTTG B317AAAACAAAAGATAATTACTTGCTCGACCTTCCGAT B318AAACAAGATGGTATCGCAAACTAAGTGACAATAAG B319AAAACTTATTGTCACTTACTTTGCGATACCATCTT B320GAGACCTTTGAGCTTCCGAGACTGGTCTCAGTTTTGGGACCATTCAAAACAG B321TGAGACCAGTCTCGGAAGCTCAAAGGTCTCGTTTTAGAGCTATGCTGTTTTG B352aaacTACTTTACGCAGCGCGGAGTTCGGTTTTTTg B353aaaacAAAAAACCGAACTCCGCGCTGCGTAAAGTA HC008_SPATGCCGGTACTGCCGGGCCTCTTGCGGGATTACGAAATCATCCTG HC009_SPGTGACTGGCGATGCTGTCGGAATGGACGATCACACTACTCTTCTT HC010_SPTTAAGAAATAATCTTCATCTAAAATATACTTCAGTCACCTCCTAGCTGAC HC011_SPATTGATTTGAGTCAGCTAGGAGGTGACTGAAGTATATTTTAGATGAAG HC014_SPGAGACCTTTGAGCTTCCGAGACTGGTCTCAGTTTTGGGACCATTCAAACAGCATAGCTCTAAAACCTCGTAGACTATTTTTGTC HC015_SPGAGACCAGTCTCGGAAGCTCAAAGGTCTCGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACTTCAGCACACTGAGACTTG L403 AGTCATCCCAGCAACAAATGG L409 CGTGGTAAATCGGATAACGTTCCAAGTGAAGL422 Tgctcttcttcacaaacaaggg L426 AAGCCAAAGTTTGGCACCACC L430GTAGCTTATTCAGTCCTAGTGG L444CGTTTGTTGAACTAATGGGTGCAAATTACGAATCTTCTCCTGACG L445CGTCAGGAGAAGATTCGTAATTTGCACCCATTAGTTCAACAAACG L446GATATTATGGAGCCTATTTTTGTGGGTTTTTAGGCATAAAACTATATG L447CATATAGTTTTATGCCTAAAAACCcACAAAAATAGGCTCCATAATATC L448ATTATTTCTTAATAACTAAAAATATGG L457 CGTgtacaattgctagcgtacggc L458GCACCGGTGATCACTAGTCCTAGG L459cctaggactagtgatcaccggtGCAAATATGAGCCAAATAAATATAT L461GCCGTACGCTAGCAATTGTACACGTTTGTTGAACTAATGGGTGC L481TTCAAATTTTCCCATTTGATTCTCC L488CCATATTTTTAGTTATTAAGAAATAATACCAGCCATCAGTCACCTCC W256AGACGATTCAATAGACAATAAGG W286GTTTTGGGACCATTCAAACAGCATAGCTCTAAAACCTCGTAGAC W287GCTATGCTGTTTTGAATGGTCCCAAAACcattattttaacacacgaggtg W288GCTATGCTGTTTTGAATGGTCCCAAAACGCACCCATTAGTTCAACAAACG W326AATTCTTTTCTTCATCATCGGTC W327 AAGAAAGAATGAAGATTGTTCATG W341GGTACTAATCAAAATAGTGAGGAGG W354 GTTTTTCAAAATCTGCGGTTGCG W355AAAAATTGAAAAAATGGTGGAAACAC W356ATTTCGTAAACGGTATCGGTTTCTTTTAAAGTTTTGGGACCATTCAAAACAGC W357TTTAAAAGAAACCGATACCGTTTACGAAATGTTTTAGAGCTATGCTGTTTTGA W365AAACGGTATCGGTTTCTTTTAAATTCAATTGTTTTGGGACCATTCAAAACAGC W366AATTGAATTTAAAAGAAACCGATACCGTTTGTTTTAGAGCTATGCTGTTTTGA W370GTTCCTTAAACCAAAACGGTATCGGTTTCTTTTAAATTC W371GAAACCGATACCGTTTTGGTTTAAGGAACAGGTAAAGGGCATTTAAC W376CGATTTCAGCCATTGCCTCGTC W377GCCTTTGACGAGGCAATGGCTGAAATCGNNNNNAAAAAGCGCAAGAAGAAATCAAC W391TCCGTACAACCCACAACCCTGCTAGTGAGCGTTTTGGGACCATTCAAAACAGC W392GCTCACTAGCAGGGTTGTGGGTTGTACGGAGTTTTAGAGCTATGCTGTTTTGA W393TTGTTGCCACTCTTCCTTCTTTC W397 CAGGGTTGTGGGTTGTTGCGATGGAGTTAACTCCCATCTCCW398 GGGAGTTAACTCCATCGCAACAACCCACAACCCTGCTAGTG W403GTGGTATCTATCGTGATGTGAC W404 TTACCGAAACGGAATTTATCTGC W405AAAGCTAGAGTTCCGCAATTGG W431 GTGGGTTGTACGGATTGAGTTAACTCCCATCTCCTTC W432GATGGGAGTTAACTCAATCCGTACAACCCACAACCCTG W433GCTTCACCTATTGCAGCACCAATTGACCACATGAAGATAG W434GTGGTCAATTGGTGCTGCAATAGGTGAAGCTAATGGTGATG W463CTGATTTGTATTAATTTTGAGACATTATGCTTCACCTTC W464GCATAATGTCTCAAAATTAATACAAATCAGTGAAATCATG W465GTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACGTGACAGTAATATCAG W466GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACGCTCACTAGCAGGGTTG W542ATACTTTACGCAGCGCGGAGTTCGGTTTTgTAGGAGTGGTAGTATATACACGAGTACAT

TABLE H Design of targeting and editing constructs used in this study(SEQ ID NOS 184, 184, 184, 185, and 186, respectively, in order ofappearance). Targeting Constructs Edition Template DNA Left PCR RightPCR Spacer sequence PAM bgaA R > A crR6Rk W256/W391 W302/L403GCTCACTAGCAGGGTTGTGGGTTGTACGGA TGG bgaA NE > AA crR6Rk W256/W391W392/L403 GCTCACTAGCAGGGTTGTGGGTTGTACGGA TGG ΔbgaA crR6Rk W256/W391W392/L403 GCTCACTAGCAGGGTTGTGGGTTGTACGGA TGG ΔsrtA crRSRc W256/B218B217/L403 TCCTAGCAGGATTTCTGATATTACTGTCAC TGG ermB Stop crR6Rk W256/W356W357/L403 TTTAAAAGAAACCGATACCGTTTACGAAAT TGG ΔsrtA ΔbgaA JEN51 (for LeftPCR) and W256/W465 W466/W403 same as the ones used for ΔsrtA and ΔbgaATGG JEN52 (for Right PCR) Editing Constructs Name of Primers used toresulting verify edited Edition Template DNA PCR A PCR B PCR C SOEingPCR strains genotype bgaA R > A R6 W403/W397 W398/W404 N/A W403/W404JEN56 W403/W404 bgaA NE > AA R6 W403/W431 W432/W433 W434/W404 W403/W404JEN60 W403/W404 ΔbgaA R6 B255/B256 B257/B258 N/A B255/B258 JEN52W393/W405 ΔsrtA R6 B230/W463 W464/B229 N/A B230/B229 JEN51 W422/W426ermB Stop JEN38 L422/W370 W371/L426 N/A L422/L426 JEN43 L457/L458 ΔsrtAΔbgaA same as the ones used for ΔsrtA and ΔbgaA JEN64 same as the onesused for ΔsrtA and ΔbgaA

Example 6 Optimization of the Guide RNA for Streptococcus pyogenes Cas9(Referred to as SpCas9)

Applicants mutated the tracrRNA and direct repeat sequences, or mutatedthe chimeric guide RNA to enhance the RNAs in cells.

The optimization is based on the observation that there were stretchesof thymines (Ts) in the tracrRNA and guide RNA, which might lead toearly transcription termination by the pol 3 promoter. ThereforeApplicants generated the following optimized sequences. OptimizedtracrRNA and corresponding optimized direct repeat are presented inpairs.

Optimized tracrRNA 1 (mutation underlined): (SEQ ID NO: 187)GGAACCATTCAtAACAGCATAGCAAGTTAtAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTOptimized direct repeat 1 (mutation underlined): (SEQ ID NO: 188)GTTaTAGAGCTATGCTGTTaTGAATGGTCCCAAAACOptimized tracRNA 2 (mutation underlined): (SEQ ID NO: 189)GGAACCATTCAAtACAGCATAGCAAGTTAAtATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTOptimized direct repeat 2 (mutation underlined): (SEQ ID NO: 190)GTaTTAGAGCTATGCTGTaTTGAATGGTCCCAAAAC

Applicants also optimized the chimeric guideRNA for optimal activity ineukaryotic cells.

Original guide RNA: (SEQ ID NO: 191)NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTOptimized chimeric guide RNA sequence 1: (SEQ ID NO: 192)NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTAGAAATAGCAAGTTAATATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTOptimized chimeric guide RNA sequence 2: (SEQ ID NO: 193)NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGCTGTTTTGGAAACAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT Optimized chimeric guide RNA sequence 3:(SEQ ID NO: 194) NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTATGCTGTATTGGAAACAATACAGCATAGCAAGTTAATATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

Applicants showed that optimized chimeric guide RNA works better asindicated in FIG. 3. The experiment was conducted by co-transfecting293FT cells with Cas9 and a U6-guide RNA DNA cassette to express one ofthe four RNA forms shown above. The target of the guide RNA is the sametarget site in the human Emx1 locus: “GTCACCTCCAATGACTAGGG (SEQ ID NO:195)”

Example 7 Optimization of Streptococcus thermophiles LMD-9 CRISPR1 Cas9(referred to as St1Cas9)

Applicants designed guide chimeric RNAs as shown in FIG. 4.

The St1Cas9 guide RNAs can undergo the same type of optimization as forSpCas9 guide RNAs, by breaking the stretches of poly thymines (Ts)

Example 8 Cas9 Diversity and Mutations

The CRISPR-Cas system is an adaptive immune mechanism against invadingexogenous DNA employed by diverse species across bacteria and archaea.The type II CRISPR-Cas9 system consists of a set of genes encodingproteins responsible for the “acquisition” of foreign DNA into theCRISPR locus, as well as a set of genes encoding the “execution” of theDNA cleavage mechanism; these include the DNA nuclease (Cas9), anon-coding transactivating cr-RNA (tracrRNA), and an array of foreignDNA-derived spacers flanked by direct repeats (crRNAs). Upon maturationby Cas9, the tracRNA and crRNA duplex guide the Cas9 nuclease to atarget DNA sequence specified by the spacer guide sequences, andmediates double-stranded breaks in the DNA near a short sequence motifin the target DNA that is required for cleavage and specific to eachCRISPR-Cas system. The type II CRISPR-Cas systems are found throughoutthe bacterial kingdom and highly diverse in Cas9 protein sequence andsize, tracrRNA and crRNA direct repeat sequence, genome organization ofthese elements, and the motif requirement for target cleavage. Onespecies may have multiple distinct CRISPR-Cas systems.

Applicants evaluated 207 putative Cas9s from bacterial speciesidentified based on sequence homology to known Cas9s and structuresorthologous to known subdomains, including the HNH endonuclease domainand the RuvC endonuclease domains [information from the Eugene Kooninand Kira Makarova]. Phylogenetic analysis based on the protein sequenceconservation of this set revealed five families of Cas9s, includingthree groups of large Cas9s (˜1400 amino acids) and two of small Cas9s(˜1100 amino acids) (FIGS. 39 and 40A-F).

In this example, Applicants show that the following mutations canconvert SpCas9 into a nicking enzyme: D10A, E762A, H840A, N854A, N863A,D986A.

Applicants provide sequences showing where the mutation points arelocated within the SpCas9 gene (FIG. 41). Applicants also show that thenickases are still able to mediate homologous recombination (Assayindicated in FIG. 2). Furthermore, Applicants show that SpCas9 withthese mutations (individually) do not induce double strand break (FIG.47).

Example 9 Supplement to DNA Targeting Specificity of the RNA-guided Cas9Nuclease

Cell Culture and Transfection

Human embryonic kidney (HEK) cell line 293FT (Life Technologies) wasmaintained in Dulbecco's modified Eagle's Medium (DMEM) supplementedwith 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (LifeTechnologies), 100U/mL penicillin, and 100 μg/mL streptomycin at 37° C.with 5% CO2 incubation.

293FT cells were seeded either onto 6-well plates, 24-well plates, or96-well plates (Corning) 24 hours prior to transfection. Cells weretransfected using Lipofectamine 2000 (Life Technologies) at 80-90%confluence following the manufacturer's recommended protocol. For eachwell of a 6-well plate, a total of 1 ug of Cas9+sgRNA plasmid was used.For each well of a 24-well plate, a total of 500 ng Cas9+sgRNA plasmidwas used unless otherwise indicated. For each well of a 96-well plate,65 ng of Cas9 plasmid was used at a 1:1 molar ratio to the U6-sgRNA PCRproduct.

Human embryonic stem cell line HUES9 (Harvard Stem Cell Institute core)was maintained in feeder-free conditions on GelTrex (Life Technologies)in mTesR medium (Stemcell Technologies) supplemented with 100 ug/mlNormocin (InvivoGen). HUES9 cells were transfected with Amaxa P3 PrimaryCell 4-D Nucleofector Kit (Lonza) following the manufacturer's protocol.

SURVEYOR Nuclease Assay for Genome Modification

293FT cells were transfected with plasmid DNA as described above. Cellswere incubated at 37° C. for 72 hours post-transfection prior to genomicDNA extraction. Genomic DNA was extracted using the QuickExtract DNAExtraction Solution (Epicentre) following the manufacturer's protocol.Briefly, pelleted cells were resuspended in QuickExtract solution andincubated at 65° C. for 15 minutes and 98° C. for 10 minutes.

The genomic region flanking the CRISPR target site for each gene was PCRamplified (primers listed in Tables J and K), and products were purifiedusing QiaQuick Spin Column (Qiagen) following the manufacturer'sprotocol. 400 ng total of the purified PCR products were mixed with 2 μl10× Taq DNA Polymerase PCR buffer (Enzymatics) and ultrapure water to afinal volume of 20 μl, and subjected to a re-annealing process to enableheteroduplex formation: 95° C. for 10 min, 95° C. to 85° C. ramping at−2° C./s, 85° C. to 25° C. at −0.25° C./s, and 25° C. hold for 1 minute.After re-annealing, products were treated with SURVEYOR nuclease andSURVEYOR enhancer S (Transgenomics) following the manufacturer'srecommended protocol, and analyzed on 4-20% Novex TBE poly-acrylamidegels (Life Technologies). Gels were stained with SYBR Gold DNA stain(Life Technologies) for 30 minutes and imaged with a Gel Doc gel imagingsystem (Bio-rad). Quantification was based on relative band intensities.

Northern Blot Analysis of tracrRNA Expression in Human Cells

Northern blots were performed as previously described 1. Briefly, RNAswere heated to 95° C. for 5 min before loading on 8% denaturingpolyacrylamide gels (SequaGel, National Diagnostics). Afterwards, RNAwas transferred to a pre-hybridized Hybond N+ membrane (GE Healthcare)and crosslinked with Stratagene UV Crosslinker (Stratagene). Probes werelabeled with [gamma-32P]ATP (Perkin Elmer) with T4 polynucleotide kinase(New England Biolabs). After washing, membrane was exposed to phosphorscreen for one hour and scanned with phosphorimager (Typhoon).

Bisulfite Sequencing to Assess DNA Methylation Status

HEK 293FT cells were transfected with Cas9 as described above. GenomicDNA was isolated with the DNeasy Blood & Tissue Kit (Qiagen) andbisulfite converted with EZ DNA Methylation-Lightning Kit (ZymoResearch). Bisulfite PCR was conducted using KAPA2G Robust HotStart DNAPolymerase (KAPA Biosystems) with primers designed using the BisulfitePrimer Seeker (Zymo Research, Tables J and K). Resulting PCR ampliconswere gel-purified, digested with EcoRI and HindIII, and ligated into apUC19 backbone prior to transformation. Individual clones were thenSanger sequenced to assess DNA methylation status.

In vitro Transcription and Cleavage Assay

HEK 293FT cells were transfected with Cas9 as described above. Wholecell lysates were then prepared with a lysis buffer (20 mM HEPES, 100 mMKCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 0.1% Triton X-100) supplementedwith Protease Inhibitor Cocktail (Roche). T7-driven sgRNA was in vitrotranscribed using custom oligos (Example 10) and HiScribe T7 In VitroTranscription Kit (NEB), following the manufacturer's recommendedprotocol. To prepare methylated target sites, pUC19 plasmid wasmethylated by M.SssI and then linearized by NheI. The in vitro cleavageassay was performed as follows: for a 20 uL cleavage reaction, 10 uL ofcell lysate with incubated with 2 uL cleavage buffer (100 mM HEPES, 500mM KCl, 25 mM MgCl2, 5 mM DTT, 25% glycerol), the in vitro transcribedRNA, and 300 ng pUC19 plasmid DNA.

Deep Sequencing to Assess Targeting Specificity

HEK 293FT cells plated in 96-well plates were transfected with Cas9plasmid DNA and single guide RNA (sgRNA) PCR cassette 72 hours prior togenomic DNA extraction (FIG. 72). The genomic region flanking the CRISPRtarget site for each gene was amplified (FIG. 74, FIG. 80, (Example 10)by a fusion PCR method to attach the Illumina P5 adapters as well asunique sample-specific barcodes to the target amplicons (schematicdescribed in FIG. 73). PCR products were purified using EconoSpin96-well Filter Plates (Epoch Life Sciences) following the manufacturer'srecommended protocol.

Barcoded and purified DNA samples were quantified by Quant-iT PicoGreendsDNA Assay Kit or Qubit 2.0 Fluorometer (Life Technologies) and pooledin an equimolar ratio. Sequencing libraries were then deep sequencedwith the Illumina MiSeq Personal Sequencer (Life Technologies).

Sequencing Data Analysis and Indel Detection

MiSeq reads were filtered by requiring an average Phred quality (Qscore) of at least 23, as well as perfect sequence matches to barcodesand amplicon forward primers. Reads from on- and off-target loci wereanalyzed by first performing Smith-Waterman alignments against ampliconsequences that included 50 nucleotides upstream and downstream of thetarget site (a total of 120 bp). Alignments, meanwhile, were analyzedfor indels from 5 nucleotides upstream to 5 nucleotides downstream ofthe target site (a total of 30 bp). Analyzed target regions werediscarded if part of their alignment fell outside the MiSeq read itself,or if matched base-pairs comprised less than 85% of their total length.

Negative controls for each sample provided a gauge for the inclusion orexclusion of indels as putative cutting events. For each sample, anindel was counted only if its quality score exceeded μ−σ, where μ wasthe mean quality-score of the negative control corresponding to thatsample and σ was the standard deviation of same. This yielded wholetarget-region indel rates for both negative controls and theircorresponding samples. Using the negative control'sper-target-region-per-read error rate, q, the sample's observed indelcount n, and its read-count R, a maximum-likelihood estimate for thefraction of reads having target-regions with true-indels, p, was derivedby applying a binomial error model, as follows.

Letting the (unknown) number of reads in a sample having target regionsincorrectly counted as having at least 1 indel be E, we can write(without making any assumptions about the number of true indels)

${Prob}\left( {{E\left. p \right)} = {\begin{pmatrix}{R\left( {1 - p} \right)} \\E\end{pmatrix}{q^{E}\left( {1 - q} \right)}^{{R{({1 - p})}} - E}}} \right.$

since R(1−p) is the number of reads having target-regions with no trueindels. Meanwhile, because the number of reads observed to have indelsis n, n=E+Rp, in other words the number of reads having target-regionswith errors but no true indels plus the number of reads whosetarget-regions correctly have indels. We can then re-write the above

${{Prob}\left( {E{p}} \right)} = {{Prob}\left( {n = {{E + {{Rp}\left. p \right)}} = {\begin{pmatrix}{R\left( {1 - P} \right)} \\{n - {Rp}}\end{pmatrix}{q^{n - {Rp}}\left( {1 - q} \right)}^{R - n}}}} \right.}$

Taking all values of the frequency of target-regions with true-indels Pto be equally probable a priori, Prob(n|p)∝Prob(p|n). Themaximum-likelihood estimate (MLE) for the frequency of target regionswith true-indels was therefore set as the value of P that maximizedProb(n|p). This was evaluated numerically.

In order to place error bounds on the true-indel read frequencies in thesequencing libraries themselves. Wilson score intervals (2) werecalculated for each sample, given the MLE-estimate for true-indeltarget-regions, Rp, and the number of reads R. Explicitly, the lowerbound l and upper bound u were calculated as

$l = {\left( {{Rp} + \frac{z^{2}}{2} - {z\sqrt{{{Rp}\left( {1 - p} \right)} + {z^{2}\text{/}4}}}} \right)\text{/}\left( {R + z^{2}} \right)}$$u = {\left( {{Rp} + \frac{z^{2}}{2} - {z\sqrt{{{Rp}\left( {1 - p} \right)} + {z^{2}\text{/}4}}}} \right)\text{/}\left( {R + z^{2}} \right)}$

where z, the standard score for the confidence required in normaldistribution of variance 1, was set to 1.96, meaning a confidence of95%. The maximum upper bounds and minimum lower bounds for eachbiological replicate are listed in FIGS. 80-83.

qRT-PCR Analysis of Relative Cas9 and sgRNA Expression

293FT cells plated in 24-well plates were transfected as describedabove. 72 hours post-transfection, total RNA was harvested with miRNeasyMicro Kit (Qiagen). Reverse-strand synthesis for sgRNAs was performedwith qScript Flex cDNA kit (VWR) and custom first-strand synthesisprimers (Tables J and K). qPCR analysis was performed with Fast SYBRGreen Master Mix (Life Technologies) and custom primers (Tables J andK), using GAPDH as an endogenous control. Relative quantification wascalculated by the ΔΔCT method.

TABLE I Target site sequences. Tested target sites forS. pyogenes type II CRISPR system with therequisite PAM. Cells were transfected withCas9 and either crRNA-tracrRNA or chimeric sgRNA for each target.Target site genomic Target site sequence ID target (5′ to 3′) PAM strand1 EMX1 GTCACCTCCAATGACTAGGG TGG + (SEQ ID NO: 319) 2 EMX1GACATCGATGTCCTCCCCAT TGG − (SEQ ID NO: 196) 3 EMX1 GAGTCCGAGCAGAAGAAGAAGGG + (SEQ ID NO: 197) 6 EMX1 GCGCCACCGGTTGATGTGAT GGG −(SEQ ID NO: 198) 10 EMX1 GGGGCACAGATGAGAAACTC AGG − (SEQ ID NO: 199) 11EMX1 GTACAAACGGCAGAAGCTGG AGG + (SEQ ID NO: 200) 12 EMX1GGCAGAAGCTGGAGGAGGAA GGG + (SEQ ID NO: 201) 13 EMX1 GGAGCCCTTCTTCTTCTGCTCGG − (SEQ ID NO: 202) 14 EMX1 GGGCAACCACAAACCCACGA GGG +(SEQ ID NO: 203) 15 EMX1 GCTCCCATCACATCAACCGG TGG + (SEQ ID NO: 204) 16EMX1 GTGGCGCATTGCCACGAAGC AGG + (SEQ ID NO: 205) 17 EMX1GGCAGAGTGCTGCTTGCTGC TGG + (SEQ ID NO: 206) 18 EMX1 GCCCCTGCGTGGGCCCAAGCTGG + (SEQ ID NO: 207) 19 EMX1 GAGTGGCCAGAGTCCAGCTT GGG −(SEQ ID NO: 208) 20 EMX1 GGCCTCCCCAAAGCCTGGCC AGG − (SEQ ID NO: 209) 4PVALB GGGGCCGAGATTGGGTGTTC AGG + (SEQ ID NO: 210) 5 PVALBGTGGCGAGAGGGGCCGAGAT TGG + (SEQ ID NO: 211) 1 SERPINB5GAGTGCCGCCGAGGCGGGGC GGG + (SEQ ID NO: 212) 2 SERPINB5GGAGTGCCGCCGAGGCGGGG CGG + (SEQ ID NO: 213) 3 SERPINB5GGAGAGGAGTGCCGCCGAGG CGG + (SEQ ID NO: 214)

TABLE J Primer sequences SURVEYOR assay genomic primer name targetprimer sequence (5′ to 3′) Sp-EMX1-F1 EMX1 AAAACCACCCTTCTCTCTGGC(SEQ ID NO: 36) Sp-EMX1-R1 EMX1 GGAGATTGGAGACACGGAGAG (SEQ ID NO: 37)Sp-EMX1-F2 EMX1 CCATCCCCTTCTGTGAATGT (SEQ ID NO: 215) Sp-EMX1-R2 EMX1GGAGATTGGAGACACGGAGA (SEQ ID NO: 216) Sp-PVALB-F PVALBCTGGAAAGCCAATGCCTGAC (SEQ ID NO: 38) Sp-PVALB-R PVALBGGCAGCAAACTCCTTGTCCT (SEQ ID NO: 39)qRT-PCR for Cas9 and sgRNA expression primer name primer sequence (5′to 3′) sgRNA AAGCACCGACTCGGTGCCAC (SEQ ID NO: 217) reverse- strandsynthesis EMX1.1 sgRNA TCACCTCCAATGACTAGGGG (SEQ ID NO: 218) qPCR FEMX1.1 sgRNA CAAGTTGATAACGGACTAGCCT (SEQ ID NO: qPCR R 219) EMX1.3 sgRNAAGTCCGAGCAGAAGAAGAAGTTT (SEQ ID NO: qPCR F 220) EMX1.3 sgRNATTTCAAGTTGATAACGGACTAGCCT (SEQ ID qPCR R NO: 221) Cas9 qPCR FAAACAGCAGATTCGCCTGGA (SEQ ID NO: 222) Cas9 qPCR RTCATCCGCTCGATGAAGCTC (SEQ ID NO: 223) GAPDH qPCR FTCCAAAATCAAGTGGGGCGA (SEQ ID NO: 224) GAPDH qPCR RTGATGACCCTTTTGGCTCCC (SEQ ID NO: 225) Bisulfite PCR and sequencingprimer name primer sequence (5′ to 3′) Bisulfite PCR FGAGGAATTCTTTTTTTGTTYGAATATGTTGGA (SERPINB5 locus)GGTTTTTTGGAAG (SEQ ID NO: 226) Bisulfite PCR RGAGAAGCTTAAATAAAAAACRACAATACTCAACC (SERPINB5 locus)CAACAACC (SEQ ID NO: 227) pUC19 sequencingCAGGAAACAGCTATGAC (SEQ ID NO: 228)

TABLE K Sequences for primers to test sgRNA architecture. Primershybridize to the reverse strand of the U6 promoter unlessotherwise indicated. The U6 priming site is in italics,the guide sequence is indicated as a stretch of Ns, the directrepeat sequence is highlighted in bold, and the tracrRNAsequence underlined. The secondary structure of each sgRNAarchitecture is shown in FIG. 43. primer name primer sequence (5′ to 3′)U6-Forward GCCTCTAGAGGTACCTGAGGGCCTATTTCCCATGATTCC (SEQ ID NO: 229)I: sgRNA(DR +12, ACCTCTAGAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTtracrRNA +85) TGATAACGGACTAGCCTTATTTTAACTTGCTATTTC TAGCTCTAAAACNNNNNNNNNNNNNNNNNNNNGGTGTTTCGTCCTTTCC ACAAG (SEQ ID NO: 230)II: sgRNA(DR +12, ACCTCTAGAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTtracrRNA +85) TGATAACGGACTAGCCTTATATTAACTTGCTATTTC TAGCTCT mut2AATACNNNNNNNNNNNNNNNNNNNNGGTGTTTCGTCCTTTCCA CAAG (SEQ ID NO: 231)III: sgRNA(DR +22, ACCTCTAGAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTtracrRNA +85) TGATAACGGACTAGCCTTATTTTAACTTGCTATGCTGTTTTGTT TCCAAAACAGCATAGCTCTAAAACNNNNNNNNNNNNNNNNNNNNGGTGTTTCGTCCTTTCCACAAG (SEQ ID NO: 232) IV: sgRNA(DRACCTCTAGAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGT +22, tracrRNA +85)TGATAACGGACTAGCCTTATATTAACTTGCTATGCTGTATTGT mut4 TTCCAATACAGCATAGCTCTAATACNNNNNNNNNNNNNNNNNNNNGGTGTTTCGTCCTTTCCACAAG (SEQ ID NO: 233)

TABLE L Target sites with alternate PAMs for testing PAMspecificity of Cas9. All target sites for PAMspecificity testing are found within the human EMX1 locus.Target site sequence (5′ to 3′) PAM AGGCCCCAGTGGCTGCTCT (SEQ ID NO: 234)NAA ACATCAACCGGTGGCGCAT (SEQ ID NO: 235) NATAAGGTGTGGTTCCAGAACC (SEQ ID NO: 236) NACCCATCACATCAACCGGTGG (SEQ ID NO: 237) NAGAAACGGCAGAAGCTGGAGG (SEQ ID NO: 238) NTAGGCAGAAGCTGGAGGAGGA (SEQ ID NO: 239) NTTGGTGTGGTTCCAGAACCGG (SEQ ID NO: 240) NTCAACCGGAGGACAAAGTACA (SEQ ID NO: 241) NTGTTCCAGAACCGGAGGACAA (SEQ ID NO: 242) NCAGTGTGGTTCCAGAACCGGA (SEQ ID NO: 243) NCTTCCAGAACCGGAGGACAAA (SEQ ID NO: 244) NCCCAGAAGCTGGAGGAGGAAG (SEQ ID NO: 245) NCGCATCAACCGGTGGCGCATT (SEQ ID NO: 246) NGAGCAGAAGCTGGAGGAGGAA (SEQ ID NO: 247) NGTCCTCCCTCCCTGGCCCAGG (SEQ ID NO: 248) NGCTCATCTGTGCCCCTCCCTC (SEQ ID NO: 249) NAAGGGAGGACATCGATGTCAC (SEQ ID NO: 250) NATCAAACGGCAGAAGCTGGAG (SEQ ID NO: 251) NACGGGTGGGCAACCACAAACC (SEQ ID NO: 252) NAGGGTGGGCAACCACAAACCC (SEQ ID NO: 253) NTAGGCTCCCATCACATCAACC (SEQ ID NO: 254) NTTGAAGGGCCTGAGTCCGAGC (SEQ ID NO: 255) NTCCAACCGGTGGCGCATTGCC (SEQ ID NO: 256) NTGAGGAGGAAGGGCCTGAGTC (SEQ ID NO: 257) NCAAGCTGGAGGAGGAAGGGCC (SEQ ID NO: 258) NCTGCATTGCCACGAAGCAGGC (SEQ ID NO: 259) NCCATTGCCACGAAGCAGGCCA (SEQ ID NO: 260) NCGAGAACCGGAGGACAAAGTA (SEQ ID NO: 261) NGATCAACCGGTGGCGCATTGC (SEQ ID NO: 262) NGTGAAGCTGGAGGAGGAAGGG (SEQ ID NO: 263) NGC

Example 10 Supplementary Sequences

All sequences are in the 5′ to 3′ direction. For U6 transcription, thestring of underlined Ts serve as the transcriptional terminator.

>U6-short tracrRNA (Streptococcus pyogenes SF370) (SEQ ID NO: 40)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC TTTTTTT (tracrRNA sequence in bold)>U6-DR-guide sequence-DR (Streptococcus pyogenes SF370) (SEQ ID NO: 54)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgc

>sgRNA containing +48 tracrRNA (Streptococcus pyogenes SF370)(SEQ ID NO: 55)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNN

>sgRNA containing +54 tracrRNA (Streptococcus pyogenes SF370)(SEQ ID NO: 56)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNN

>sgRNA containing +67 tracrRNA (Streptococcus pyogenes SF370)(SEQ ID NO: 57)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNN

>sgRNA containing +85 tracrRNA (Streptococcus pyogenes SF370)(SEQ ID NO: 58)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNN

>CBh-NLS-SpCas9-NLS (SEQ ID NO: 59)CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGaccggtgccaccATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCACAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACTTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTAGCA GCAGGACGCTTTAA(NLS-hSpCas9-NLS is highlighted in bold) >Sequencing amplicon for EMX1 guides 1.1, 1.14, 1.17(SEQ ID NO: 264) CCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCAACCACAAACCCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCAC  >Sequencing amplicon for EMX1 guides 1.2, 1.16(SEQ ID NO: 265) CGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCAACCACAAACCCACGAG >Sequencing amplicon for EMX1 guides 1.3, 1.13, 1.15(SEQ ID NO: 266) GGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGAT  >Sequencing amplicon for EMX1 guides 1.6(SEQ ID NO: 267) AGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGTGG >Sequencing amplicon for EMX1 guides 1.10(SEQ ID NO: 268) CCTCAGTCTTCCCATCAGGCTCTCAGCTCAGCCTGAGTGTTGAGGCCCCAGTGGCTGCTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCA >Sequencing amplicon for EMX1 guides 1.11, 1.12(SEQ ID NO: 269) TCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACA >Sequencing amplicon for EMX1 guides 1.18, 1.19(SEQ ID NO: 270) CTCCAATGACTAGGGTGGGCAACCACAAACCCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAGT >Sequencing amplicon for EMX1 guides 1.20(SEQ ID NO: 271) CTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAGTCATGGCCCCACAGGGCTTGAAGCCCGGGGCCGCCATTGACAGAG >T7 promoter F primer for annealing with target strand(SEQ ID NO: 272)GAAATTAATACGACTCACTATAGGG  >oligo containing pUC19 target site 1 for methylation (T7 reverse)(SEQ ID NO: 273) AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACAACGACGAGCGTGACACCACCCTATAGTGAGTCGTATTAATTTC >oligo containing pUC19 target site 2 for methylation (T7 reverse)(SEQ ID NO: 274) AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACGCAACAATTAATAGACTGGACCTATAGTGAGTCGTATTAATTTC

Example 11 Oligo-mediated Cas9-induced Homologous Recombination

The oligo homologous recombination test is a comparison of efficiencyacross different Cas9 variants and different HR template (oligo vs.plasmid).

293FT cells were used. SpCas9=Wildtype Cas9 and SpCas9n=nickase Cas9(D10A). The chimeric RNA target is the same EMX1 Protospacer Target 1 asin Examples 5, 9 and 10 and oligos synthesized by IDT using PAGEpurification.

FIG. 44 depicts a design of the oligo DNA used as HomologousRecombination (HR) template in this experiment. Long oligos contain 100bp homology to the EMX1 locus and a HindIII restriction site. 293FTcells were co-transfected with: first, a plasmid containing a chimericRNA targeting human EMX1 locus and wild-type cas9 protein, and second,the oligo DNA as HR template. Samples are from 293FT cells collected 96hours post transfection with Lipofectamine 2000. All products wereamplified with an EMX1 HR Primer, gel purified, followed by digestionwith HindIII to detect the efficiency of integration of HR template intothe human genome.

FIGS. 45 and 46 depict a comparison of HR efficiency induced bydifferent combination of Cas9 protein and HR template. The Cas9construct used were either wild-type Cas9 or the nickase version of Cas9(Cas9n). The HR template used were: antisense oligo DNA (Antisense-Oligoin above figure), or sense oligo DNA (Sense-Oligo in above figure), orplasmid HR template (HR template in above figure). The sense/anti-sensedefinition is that the actively-transcribed strand with sequencecorresponding to the transcribed mRNA is defined as the sense strand ofgenome. HR Efficiency is shown as percentage of HindIII digestion bandas against all genomic PCR amplified product (bottom numbers).

Example 12 Autistic Mouse

Recent large-scale sequencing initiatives have produced a large numberof genes associated with disease. Discovering the genes is only thebeginning in understanding what the gene does and how it leads to adiseased phenotype. Current technologies and approaches to studycandidate genes are slow and laborious. The gold standards, genetargeting and genetic knockouts, require a significant investment intime and resources, both monetary and in terms of research personnel.Applicants set out to utilize the hSpCas9 nuclease to target many genesand do so with higher efficiency and lower turnaround compared to anyother technology. Because of the high efficiency of hSpCas9 Applicantscan do RNA injection into mouse zygotes and immediately getgenome-modified animals without the need to do any preliminary genetargeting in mESCs.

Chromodomain helicase DNA binding protein 8 (CHD8) is a pivotal gene ininvolved in early vertebrate development and morphogenesis. Mice lackingCHD8 die during embryonic development. Mutations in the CHD8 gene havebeen associated with autism spectrum disorder in humans. Thisassociation was made in three different papers published simultaneouslyin Nature. The same three studies identified a plethora of genesassociated with autism spectrum disorder. Applicants' aim was to createknockout mice for the four genes that were found in all papers, Chd8,Katna12, Kctd13, and Scn2a. In addition, Applicants chose two othergenes associated with autism spectrum disorder, schizophrenia, and ADHD,GIT1, CACNA1C, and CACNB2. And finally, as a positive control Applicantsdecide to target MeCP2.

For each gene Applicants designed three gRNAs that would likely knockoutthe gene. A knockout would occur after the hSpCas9 nuclease makes adouble strand break and the error prone DNA repair pathway,non-homologous end joining, corrects the break, creating a mutation. Themost likely result is a frameshift mutation that would knockout thegene. The targeting strategy involved finding proto-spacers in the exonsof the gene that had a PAM sequence, NGG, and was unique in the genome.Preference was given to proto-spacers in the first exon, which would bemost deleterious to the gene.

Each gRNA was validated in the mouse cell line. Neuro-N2a, by liposomaltransient co-transfection with hSpCas9. 72 hours post-transfectiongenomic DNA was purified using QuickExtract DNA from Epicentre. PCR wasperformed to amplify the locus of interest. Subsequently the SURVEYORMutation Detection Kit from Transgenomics was followed. The SURVEYORresults for each gRNA and respective controls are shown in FIG. 65. Apositive SURVEYOR result is one large band corresponding to the genomicPCR and two smaller bands that are the product of the SURVEYOR nucleasemaking a double-strand break at the site of a mutation. The averagecutting efficiency of each gRNA was also determined for each gRNA. ThegRNA that was chosen for injection was the highest efficiency gRNA thatwas the most unique within the genome.

RNA (hSpCas9+gRNA RNA) was injected into the pronucleus of a zygote andlater transplanted into a foster mother. Mothers were allowed to go fullterm and pups were sampled by tail snip 10 days postnatal. DNA wasextracted and used as a template for PCR, which was then processed bySURVEYOR. Additionally, PCR products were sent for sequencing. Animalsthat were detected as being positive in either the SURVEYOR assay or PCRsequencing would have their genomic PCR products cloned into a pUC19vector and sequenced to determine putative mutations from each allele.

So far, mice pups from the Chd8 targeting experiment have been fullyprocessed up to the point of allele sequencing. The Surveyor results for38 live pups (lanes 1-38) 1 dead pup (lane 39) and 1 wild-type pup forcomparison (lane 40) are shown in FIG. 66. Pups 1-19 were injected withgRNA Chd8.2 and pups 20-38 were injected with gRNA Chd8.3. Of the 38live pups, 13 were positive for a mutation. The one dead pup also had amutation. There was no mutation detected in the wild-type sample.Genomic PCR sequencing was consistent with the SURVEYOR assay findings.

Example 13 CRISPR/Cas-Mediated Transcriptional Modulation

FIG. 67 depicts a design of the CRISPR-TF (Transcription Factor) withtranscriptional activation activity. The chimeric RNA is expressed by U6promoter, while a human-codon-optimized, double-mutant version of theCas9 protein (hSpCas9m), operably linked to triple NLS and a VP64functional domain is expressed by a EF1a promoter. The double mutations,D10A and H840A, renders the cas9 protein unable to introduce anycleavage but maintained its capacity to bind to target DNA when guidedby the chimeric RNA.

FIG. 68 depicts transcriptional activation of the human SOX2 gene withCRISPR-TF system (Chimeric RNA and the Cas9-NLS-VP64 fusion protein).293FT cells were transfected with plasmids bearing two components: (1)U6-driven different chimeric RNAs targeting 20-bp sequences within oraround the human SOX2 genomic locus, and (2) EF1a-driven hSpCas9m(double mutant)-NLS-VP64 fusion protein. 96 hours post transfection,293FT cells were harvested and the level of activation is measured bythe induction of mRNA expression using a qRT-PCR assay. All expressionlevels are normalized against the control group (grey bar), whichrepresents results from cells transfected with the CRISPR-TF backboneplasmid without chimeric RNA. The qRT-PCR probes used for detecting theSOX2 mRNA is Taqman Human Gene Expression Assay (Life Technologies). Allexperiments represents data from 3 biological replicates, n=3, errorbars show s.e.m.

Example 14 NLS: Cas9 NLS

293FT cells were transfected with plasmid containing two components: (1)EF1a promoter driving the expression of Cas9 (wild-typehuman-codon-optimized Sp Cas9) with different NLS designs (2) U6promoter driving the same chimeric RNA targeting human EMX1 locus.

Cells were collect at 72h time point post transfection, and thenextracted with 50 μl of the QuickExtract genomic DNA extraction solutionfollowing manufacturer's protocol. Target EMX1 genomic DNA were PCRamplified and then Gel-purify with 1% agarose gel. Genomic PCR productwere re-anneal and subjected to the Surveyor assay followingmanufacturer's protocol. The genomic cleavage efficiency of differentconstructs were measured using SDS-PAGE on a 4-12% TBE-PAGE gel (LifeTechnologies), analyzed and quantified with ImageLab (Bio-rad) software,all following manufacturer's protocol.

FIG. 69 depicts a design of different Cas9 NLS constructs. All Cas9 werethe human-codon-optimized version of the Sp Cas9. NLS sequences arelinked to the cas9 gene at either N-terminus or C-terminus. All Cas9variants with different NLS designs were cloned into a backbone vectorcontaining so it is driven by EF1a promoter. On the same vector there isa chimeric RNA targeting human EMX1 locus driven by U6 promoter,together forming a two-component system.

TABLE M Cas9 NLS Design Test Results. Quantification of genomic cleavageof different cas9-nls constructs by surveyor assay. Percentage GenomeCleavage Biological Biological Biological Error (S.E.M., as measured byReplicate 1 Replicate 2 Replicate 3 standard error Surveyor assay (%)(%) (%) Average (%) of the mean) Cas9 (No NLS) 2.50 3.30 2.73 2.84 0.24Cas9 with N-term 7.61 6.29 5.46 6.45 0.63 NLS Cas9 with C-term 5.75 4.864.70 5.10 0.33 NLS Cas9 with Double 9.08 9.85 7.78 8.90 0.60 (N-term andC- term) NLS

FIG. 70 depicts the efficiency of genomic cleavage induced by Cas9variants bearing different NLS designs. The percentage indicate theportion of human EMX1 genomic DNA that were cleaved by each construct.All experiments are from 3 biological replicates. n=3, error indicatesS.E.M.

Example 15 Engineering of Microalgae Using Cas9

Methods of delivering Cas9

Method 1: Applicants deliver Cas9 and guide RNA using a vector thatexpresses Cas9 under the control of a constitutive promoter such asHsp70A-Rbc S2 or Beta2-tubulin. Method 2: Applicants deliver Cas9 and T7polymerase using vectors that expresses Cas9 and T7 polymerase under thecontrol of a constitutive promoter such as Hsp70A-Rbc S2 orBeta2-tubulin. Guide RNA will be delivered using a vector containing T7promoter driving the guide RNA.

Method 3: Applicants deliver Cas9 mRNA and in vitro transcribed guideRNA to algae cells. RNA can be in vitro transcribed. Cas9 mRNA willconsist of the coding region for Cas9 as well as 3′UTR from Cop1 toensure stabilization of the Cas9 mRNA.

For Homologous recombination, Applicants provide an additional homologydirected repair template.

Sequence for a cassette driving the expression of Cas9 under the control of beta-2tubulin promoter, followed by the 3′ UTR of Cop1. (SEQ ID NO: 275)TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACTSequence for a cassette driving the expression of T7 polymerase under the control ofbeta-2 tubulin promoter, followed by the 3′ UTR of Cop1.(SEQ ID NO: 276) TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACatgcctaagaagaagaggaaggttaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgacaaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtacgattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaaGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACTSequence of guide RNA driven by the T7 promoter (T7 promoter, Ns representtargeting sequence): (SEQ ID NO: 277) gaaatTAATACGACTCACTATANNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttt

Gene Delivery:

Chlamydomonas reinhardtii strain CC-124 and CC-125 from theChlamydomonas Resource Center will be used for electroporation.Electroporation protocol follows standard recommended protocol from theGeneArt Chlamydomonas Engineering kit.

Also, Applicants generate a line of Chlamydomonas reinhardtii thatexpresses Cas9 constitutively. This can be done by using pChlamyl(linearized using PvuI) and selecting for hygromycin resistant colonies.Sequence for pChlamyl containing Cas9 is below. In this way to achievegene knockout one simply needs to deliver RNA for the guideRNA. Forhomologous recombination Applicants deliver guideRNA as well as alinearized homologous recombination template.

pChlamyl-Cas9: (SEQ ID NO: 278)TGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGTTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGTCGCTGAGGCTTGACATGATTGGTGCGTATGTTTGTATGAAGCTACAGGACTGATTTGGCGGGCTATGAGGGCGGGGGAAGCTCTGGAAGGGCCGCGATGGGGCGCGCGGCGTCCAGAAGGCGCCATACGGCCCGCTGGCGGCACCCATCCGGTATAAAAGCCCGCGACCCCGAACGGTGACCTCCACTTTCAGCGACAAACGAGCACTTATACATACGCGACTATTCTGCCGCTATACATAACCACTCAGCTAGCTTAAGATCCCATCAAGCTTGCATGCCGGGCGCGCCAGAAGGAGCGCAGCCAAACCAGGATGATGTTTGATGGGGTATTTGAGCACTTGCAACCCTTATCCGGAAGCCCCCTGGCCCACAAAGGCTAGGCGCCAATGCAAGCAGTTCGCATGCAGCCCCTGGAGCGGTGCCCTCCTGATAAACCGGCCAGGGGGCCTATGTTCTTTACTTTTTTACAAGAGAAGTCACTCAACATCTTAAAATGGCCAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGGAGATTCGAGGTACCATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAACATATGATTCGAATGTCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGACACAAGAATCCCTGTTACTTCTCGACCGTATTGATTCGGATGATTCCTACGCGAGCCTGCGGAACGACCAGGAATTCTGGGAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGCCGCTGGCCCGCCGAGCCCTGGAGGAGCTCGGGCTGCCGGTGCCGCCGGTGCTGCGGGTGCCCGGCGAGAGCACCAACCCCGTACTGGTCGGCGAGCCCGGCCCGGTGATCAAGCTGTTCGGCGAGCACTGGTGCGGTCCGGAGAGCCTCGCGTCGGAGTCGGAGGCGTACGCGGTCCTGGCGGACGCCCCGGTGCCGGTGCCCCGCCTCCTCGGCCGCGGCGAGCTGCGGCCCGGCACCGGAGCCTGGCCGTGGCCCTACCTGGTGATGAGCCGGATGACCGGCACCACCTGGCGGTCCGCGATGGACGGCACGACCGACCGGAACGCGCTGCTCGCCCTGGCCCGCGAACTCGGCCGGGTGCTCGGCCGGCTGCACAGGGTGCCGCTGACCGGGAACACCGTGCTCACCCCCCATTCCGAGGTCTTCCCGGAACTGCTGCGGGAACGCCGCGCGGCGACCGTCGAGGACCACCGCGGGTGGGGCTACCTCTCGCCCCGGCTGCTGGACCGCCTGGAGGACTGGCTGCCGGACGTGGACACGCTGCTGGCCGGCCGCGAACCCCGGTTCGTCCACGGCGACCTGCACGGGACCAACATCTTCGTGGACCTGGCCGCGACCGAGGTCACCGGGATCGTCGACTTCACCGACGTCTATGCGGGAGACTCCCGCTACAGCCTGGTGCAACTGCATCTCAACGCCTTCCGGGGCGACCGCGAGATCCTGGCCGCGCTGCTCGACGGGGCGCAGTGGAAGCGGACCGAGGACTTCGCCCGCGAACTGCTCGCCTTCACCTTCCTGCACGACTTCGAGGTGTTCGAGGAGACCCCGCTGGATCTCTCCGGCTTCACCGATCCGGAGGAACTGGCGCAGTTCCTCTGGGGGCCGCCGGACACCGCCCCCGGCGCCTGATAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT

For all modified Chlamydomonas reinhardtii cells, Applicants used PCR,SURVEYOR nuclease assay, and DNA sequencing to verify successfulmodification.

Example 16 Use of Cas9 as a transcriptional repressor in bacteria

The ability to artificially control transcription is essential both tothe study of gene function and to the construction of synthetic genenetworks with desired properties. Applicants describe here the use ofthe RNA-guided Cas9 protein as a programmable transcriptional repressor.

Applicants have previously demonstrated how the Cas9 protein ofStreptococcus pyogenes SF370 can be used to direct genome editing inStreptococcus pneumoniae. In this study Applicants engineered the crR6Rkstrain containing a minimal CRISPR system, consisting of cas9, thetracrRNA and a repeat. The D10A-H840 mutations were introduced into cas9in this strain, giving strain crR6Rk**. Four spacers targeting differentpositions of the bgaA β-galactosidase gene promoter were cloned in theCRISPR array carried by the previously described pDB98 plasmid.Applicants observed a X to Y fold reduction in β-galactosidase activitydepending on the targeted position, demonstrating the potential of Cas9as a programmable repressor (FIG. 73).

To achieve Cas9** repression in Escherichia coli a green fluorescenceprotein (GFP) reporter plasmid (pDB127) was constructed to express thegfpmut2 gene from a constitutive promoter. The promoter was designed tocarry several NPP PAMs on both strands, to measure the effect of Cas9**binding at various positions. Applicants introduced the D10A-H840mutations into pCas9, a plasmid described carrying the tracrRNA, cas9and a minimal CRISPR array designed for the easy cloning of new spacers.Twenty-two different spacers were designed to target different regionsof the gfpmut2 promoter and open reading frame. An approximately 20-foldreduction of fluorescence of was observed upon targeting regionsoverlapping or adjacent to the −35 and −10 promoter elements and to theShine-Dalgarno sequence. Targets on both strands showed similarrepression levels. These results suggest that the binding of Cas9** toany position of the promoter region prevents transcription initiation,presumably through steric inhibition of RNAP binding.

To determine whether Cas9** could prevent transcription elongation,Applicants directed it to the reading frame of gpfmut2. A reduction influorescence was observed both when the coding and non-coding strandswhere targeted, suggesting that Cas9 binding is actually strong enoughto represent an obstacle to the running RNAP. However, while a 40%reduction in expression was observed when the coding strand was thetarget, a 20-fold reduction was observed for the non-coding strand (FIG.21b , compare T9, T10 and T11 to B9, B10 and B11). To directly determinethe effects of Cas9** binding on transcription. Applicants extracted RNAfrom strains carrying either the T5, T10, B10 or a control constructthat does not target pDB127 and subjected it to Northern blot analysisusing either a probe binding before (B477) or after (B510) the B10 andT10 target sites. Consistent with Applicants' fluorescence methods, nogfpmut2 transcription was detected when Cas9** was directed to thepromoter region (T5 target) and a transcription was observed after thetargeting of the T10 region. Interestingly, a smaller transcript wasobserved with the B477 probe. This band corresponds to the expected sizeof a transcript that would be interrupted by Cas9**, and is a directindication of a transcriptional termination caused by dgRNA::Cas9**binding to the coding strand. Surprisingly, Applicants detected notranscript when the non-coding strand was targeted (B10). Since Cas9**binding to the B10 region is unlikely to interfere with transcriptioninitiation, this result suggests that the mRNA was degraded. DgRNA::Cas9was shown to bind ssRNA in vitro. Applicants speculate that binding maytrigger degradation of the mRNA by host nucleases. Indeed, ribosomestalling can induce cleavage on the translated mRNA in E. coli.

Some applications require a precise tuning gene expression rather thanits complete repression. Applicants sought to achieve intermediaterepression levels through the introduction of mismatches that willweaken the crRNA/target interactions. Applicants created a series ofspacers based on the B1, T5 and B10 constructs with increasing numbersof mutations in the 5′ end of the crRNA. Up to 8 mutations in B1 and T5did not affect the repression level, and a progressive increased influorescence was observed for additional mutations.

The observed repression with only an 8 nt match between the crRNA andits target raises the question of off-targeting effects of the use ofCas9** as a transcriptional regulator. Since a good PAM (NGG) is alsorequired for Cas9 binding, the number of nucleotides to match to obtainsome level of respiration is 10. A 10 nt match occurs randomly onceevery ˜1 Mbp, and such sites are thus likely to be found even in smallbacterial genomes. However, to effectively repress transcription, suchsite needs to be in the promoter region of gene, which makesoff-targeting much less likely. Applicants also showed that geneexpression can be affected if the non-coding strand of a gene istargeted. For this to happen, a random target would have to be in theright orientation, but such events relatively more likely to happen. Asa matter of fact, during the course of this study Applicants were unableto construct one of the designed spacer on pCas9**. Applicants laterfound this spacer showed a 12 bp match next to a good PAM in theessential murC gene. Such off-targeting could easily be avoided by asystematic blast of the designed spacers.

Aspects of the invention are further described in the following numberedparagraphs:

1. A vector system comprising one or more vectors, wherein the systemcomprises

a. a first regulatory element operably linked to a traer mate sequenceand one or more insertion sites for inserting a guide sequence upstreamof the traer mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aCRISPR enzyme complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the traer mate sequence that ishybridized to the traer sequence; and

b. a second regulatory element operably linked to an enzyme-codingsequence encoding said CRISPR enzyme comprising a nuclear localizationsequence;

wherein components (a) and (b) are located on the same or differentvectors of the system.

2. The vector system of paragraph 1, wherein component (a) furthercomprises the traer sequence downstream of the traer mate sequence underthe control of the first regulatory element.

3. The vector system of paragraph 1, wherein component (a) furthercomprises two or more guide sequences operably linked to the firstregulatory element, wherein when expressed, each of the two or moreguide sequences direct sequence specific binding of a CRISPR complex toa different target sequence in a eukaryotic cell.

4. The vector system of paragraph 1, wherein the system comprises thetraer sequence under the control of a third regulatory element.

5. The vector system of paragraph 1, wherein the traer sequence exhibitsat least 50% of sequence complementarity along the length of the traermate sequence when optimally aligned.

6. The vector system of paragraph 1, wherein the CRISPR enzyme comprisesone or more nuclear localization sequences of sufficient strength todrive accumulation of said CRISPR enzyme in a detectable amount in thenucleus of a eukaryotic cell.

7. The vector system of paragraph 1, wherein the CRISPR enzyme is a typeII CRISPR system enzyme.

8. The vector system of paragraph 1, wherein the CRISPR enzyme is a Cas9enzyme.

9. The vector system of paragraph 1, wherein the CRISPR enzyme iscodon-optimized for expression in a eukaryotic cell.

10. The vector system of paragraph 1, wherein the CRISPR enzyme directscleavage of one or two strands at the location of the target sequence.

11. The vector system of paragraph 1, wherein the CRISPR enzyme lacksDNA strand cleavage activity.

12. The vector system of paragraph 1, wherein the first regulatoryelement is a polymerase III promoter.

13. The vector system of paragraph 1, wherein the second regulatoryelement is a polymerase II promoter.

14. The vector system of paragraph 4, wherein the third regulatoryelement is a polymerase Ill promoter.

15. The vector system of paragraph 1, wherein the guide sequence is atleast 15 nucleotides in length.

16. The vector system of paragraph 1, wherein fewer than 50% of thenucleotides of the guide sequence participate in self-complementarybase-pairing when optimally folded.

17. A vector comprising a regulatory element operably linked to anenzyme-coding sequence encoding a CRISPR enzyme comprising one or morenuclear localization sequences, wherein said regulatory element drivestranscription of the CRISPR enzyme in a eukaryotic cell such that saidCRISPR enzyme accumulates in a detectable amount in the nucleus of theeukaryotic cell.

18. The vector of paragraph 17, wherein said regulatory element is apolymerase II promoter.

19. The vector of paragraph 17, wherein said CRISPR enzyme is a typeIICRISPR system enzyme.

20. The vector of paragraph 17, wherein said CRISPR enzyme is a Cas9enzyme.

21. The vector of paragraph 17, wherein said CRISPR enzyme lacks theability to cleave one or more strands of a target sequence to which itbinds.

22. A CRISPR enzyme comprising one or more nuclear localizationsequences of sufficient strength to drive accumulation of said CRISPRenzyme in a detectable amount in the nucleus of a eukaryotic cell.

23. The CRISPR enzyme of paragraph 22, wherein said CRISPR enzyme is atype IICRISPR system enzyme.

24. The CRISPR enzyme of paragraph 22, wherein said CRISPR enzyme is aCas9 enzyme.

25. The CRISPR enzyme of paragraph 22, wherein said CRISPR enzyme lacksthe ability to cleave one or more strands of a target sequence to whichit binds.

26. A eukaryotic host cell comprising:

a. a first regulatory element operably linked to a traer mate sequenceand one or more insertion sites for inserting a guide sequence upstreamof the traer mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aCRISPR enzyme complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the traer mate sequence that ishybridized to the traer sequence; and/or

b. a second regulatory element operably linked to an enzyme-codingsequence encoding said CRISPR enzyme comprising a nuclear localizationsequence.

27. The eukaryotic host cell of paragraph 26, wherein said host cellcomprises components (a) and (b).

28. The eukaryotic host cell of paragraph 26, wherein component (a),component (b), or components (a) and (b) are stably integrated into agenome of the host eukaryotic cell.

29. The eukaryotic host cell of paragraph 26, wherein component (a)further comprises the traer sequence downstream of the traer matesequence under the control of the first regulatory element.

30. The eukaryotic host cell of paragraph 26, wherein component (a)further comprises two or more guide sequences operably linked to thefirst regulatory element, wherein when expressed, each of the two ormore guide sequences direct sequence specific binding of a CRISPRcomplex to a different target sequence in a eukaryotic cell.

31. The eukaryotic host cell of paragraph 26, further comprising a thirdregulatory element operably linked to said traer sequence.

32. The eukaryotic host cell of paragraph 26, wherein the traer sequenceexhibits at least 50% of sequence complementarity along the length ofthe traer mate sequence when optimally aligned.

33. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzymecomprises one or more nuclear localization sequences of sufficientstrength to drive accumulation of said CRISPR enzyme in a detectablemount in the nucleus of a eukaryotic cell.

34. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzymeis a type II CRISPR system enzyme.

35. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzymeis a Cas9 enzyme.

36. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzymeis codon-optimized for expression in a eukaryotic cell.

37. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzymedirects cleavage of one or two strands at the location of the targetsequence.

38. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzymelacks DNA strand cleavage activity.

39. The eukaryotic host cell of paragraph 26, wherein the firstregulatory element is a polymerase III promoter.

40. The eukaryotic host cell of paragraph 26, wherein the secondregulatory element is a polymerase II promoter.

41. The eukaryotic host cell of paragraph 31, wherein the thirdregulatory element is a polymerase III promoter.

42. The eukaryotic host cell of paragraph 26, wherein the guide sequenceis at least 15 nucleotides in length.

43. The eukaryotic host cell of paragraph 26, wherein fewer than 50% ofthe nucleotides of the guide sequence participate in self-complementarybase-pairing when optimally folded.

44. A non-human animal comprising a eukaryotic host cell of any one ofparagraphs 26-43.

45. A kit comprising a vector system and instructions for using saidkit, the vector system comprising:

a. a first regulatory element operably linked to a traer mate sequenceand one or more insertion sites for inserting a guide sequence upstreamof the traer mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aCRISPR enzyme complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the traer mate sequence that ishybridized to the traer sequence; and/or

b. a second regulatory element operably linked to an enzyme-codingsequence encoding said CRISPR enzyme comprising a nuclear localizationsequence.

46. The kit of paragraph 45, wherein said kit comprises components (a)and (b) located on the same or different vectors of the system.

47. The kit of paragraph 45, wherein component (a) further comprises thetraer sequence downstream of the traer mate sequence under the controlof the first regulatory element.

48. The kit of paragraph 45, wherein component (a) further comprises twoor more guide sequences operably linked to the first regulatory element,wherein when expressed, each of the two or more guide sequences directsequence specific binding of a CRISPR complex to a different targetsequence in a eukaryotic cell.

49. The kit of paragraph 45, wherein the system comprises the traersequence under the control of a third regulatory element.

50. The kit of paragraph 45, wherein the traer sequence exhibits atleast 50% of sequence complementarity along the length of the traer matesequence when optimally aligned.

51. The kit of paragraph 45, wherein the CRISPR enzyme comprises one ormore nuclear localization sequences of sufficient strength to driveaccumulation of said CRISPR enzyme in a detectable mount in the nucleusof a eukaryotic cell.

52. The kit of paragraph 45, wherein the CRISPR enzyme is a type IICRISPR system enzyme.

53. The kit of paragraph 45, wherein the CRISPR enzyme is a Cas9 enzyme.

54. The kit of paragraph 45, wherein the CRISPR enzyme iscodon-optimized for expression in a eukaryotic cell.

55. The kit of paragraph 45, wherein the CRISPR enzyme directs cleavageof one or two strands at the location of the target sequence.

56. The kit of paragraph 45, wherein the CRISPR enzyme lacks DNA strandcleavage activity.

57. The kit of paragraph 45, wherein the first regulatory element is apolymerase III promoter.

58. The kit of paragraph 45, wherein the second regulatory element is apolymerase 11 promoter.

59. The kit of paragraph 49, wherein the third regulatory element is apolymerase III promoter.

60. The kit of paragraph 45, wherein the guide sequence is at least 15nucleotides in length.

61. The kit of paragraph 45, wherein fewer than 50% of the nucleotidesof the guide sequence participate in self-complementary base-pairingwhen optimally folded.

62. A computer system for selecting a candidate target sequence within anucleic acid sequence in a eukaryotic cell for targeting by a CRISPRcomplex, the system comprising:

a. a memory unit configured to receive and/or store said nucleic acidsequence; and

b. one or more processors alone or in combination programmed to (i)locate a CRISPR motif sequence within said nucleic acid sequence, and(ii) select a sequence adjacent to said located CRISPR motif sequence asthe candidate target sequence to which the CRISPR complex binds.

63. The computer system of paragraph 62, wherein said locating stepcomprises identifying a CRISPR motif sequence located less than about500 nucleotides away from said target sequence.

64. The computer system of paragraph 62, wherein said candidate targetsequence is at least 10 nucleotides in length.

65. The computer system of paragraph 62, wherein the nucleotide at the3′ end of the candidate target sequence is located no more than about 10nucleotides upstream of the CRISPR motif sequence.

66. The computer system of paragraph 62, wherein the nucleic acidsequence in the eukaryotic cell is endogenous to the eukaryotic genome.

67. The computer system of claim 62, wherein the nucleic acid sequencein the eukaryotic cell is exogenous to the eukaryotic genome.

68. A computer-readable medium comprising codes that, upon execution byone or more processors, implements a method of selecting a candidatetarget sequence within a nucleic acid sequence in a eukaryotic cell fortargeting by a CRISPR complex, said method comprising: (a) locating aCRISPR motif sequence within said nucleic acid sequence, and (b)selecting a sequence adjacent to said located CRISPR motif sequence asthe candidate target sequence to which the CRISPR complex binds.

69. The computer-readable medium of paragraph 68, wherein said locatingcomprises locating a CRISPR motif sequence that is less than about 500nucleotides away from said target sequence.

70. The computer-readable of paragraph 68, wherein said candidate targetsequence is at least 10 nucleotides in length.

71. The computer-readable of paragraph 68, wherein the nucleotide at the3′ end of the candidate target sequence is located no more than about 10nucleotides upstream of the CRISPR motif sequence.

72. The computer-readable of paragraph 68, wherein the nucleic acidsequence in the eukaryotic cell is endogenous the eukaryotic genome.

73. The computer-readable of paragraph 68, wherein the nucleic acidsequence in the eukaryotic cell is exogenous to the eukaryotic genome.

74. A method of modifying a target polynucleotide in a eukaryotic cell,the method comprising allowing a CRISPR complex to bind to the targetpolynucleotide to effect cleavage of said target polynucleotide therebymodifying the target polynucleotide, wherein the CRISPR complexcomprises a CRISPR enzyme complexed with a guide sequence hybridized toa target sequence within said target polynucleotide, wherein said guidesequence is linked to a traer mate sequence which in turn hybridizes toa traer sequence.

75. The method of paragraph 74, wherein said cleavage comprises cleavingone or two strands at the location of the target sequence by said CRISPRenzyme.

76. The method of paragraph 74, wherein said cleavage results indecreased transcription of a target gene.

77. The method of paragraph 74, further comprising repairing saidcleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide.

78. The method of paragraph 77, wherein said mutation results in one ormore amino acid changes in a protein expressed from a gene comprisingthe target sequence.

79. The method of paragraph 74, further comprising delivering one ormore vectors to said eukaryotic cell, wherein the one or more vectorsdrive expression of one or more of: the CRISPR enzyme, the guidesequence linked to the traer mate sequence, and the traer sequence.

80. The method of paragraph 79, wherein said vectors are delivered tothe eukaryotic cell in a subject.

81. The method of paragraph 74, wherein said modifying takes place insaid eukaryotic cell in a cell culture.

82. The method of paragraph 74, further comprising isolating saideukaryotic cell from a subject prior to said modifying.

83. The method of paragraph 82, further comprising returning saideukaryotic cell and/or cells derived therefrom to said subject.

84. A method of modifying expression of a polynucleotide in a eukaryoticcell, the method comprising: allowing a CRISPR complex to bind to thepolynucleotide such that said binding results in increased or decreasedexpression of said polynucleotide; wherein the CRISPR complex comprisesa CRISPR enzyme complexed with a guide sequence hybridized to a targetsequence within said polynucleotide, wherein said guide sequence islinked to a traer mate sequence which in turn hybridizes to a traersequence.

85. The method of paragraph 74, further comprising delivering one ormore vectors to said eukaryotic cells, wherein the one or more vectorsdrive expression of one or more of: the CRISPR enzyme, the guidesequence linked to the traer mate sequence, and the traer sequence.

86. A method of generating a model eukaryotic cell comprising a mutateddisease gene, the method comprising:

a. introducing one or more vectors into a eukaryotic cell, wherein theone or more vectors drive expression of one or more of: a CRISPR enzyme,a guide sequence linked to a traer mate sequence, and a traer sequence;and

b. allowing a CRISPR complex to bind to a target polynucleotide toeffect cleavage of the target polynucleotide within said disease gene,wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridized to the target sequence withinthe target polynucleotide, and (2) the traer mate sequence that ishybridized to the traer sequence, thereby generating a model eukaryoticcell comprising a mutated disease gene.

87. The method of paragraph 86, wherein said cleavage comprises cleavingone or two strands at the location of the target sequence by said CRISPRenzyme.

88. The method of paragraph 86, wherein said cleavage results indecreased transcription of a target gene.

89. The method of paragraph 86, further comprising repairing saidcleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide.

90. The method of paragraph 89, wherein said mutation results in one ormore amino acid changes in a protein expressed from a gene comprisingthe target sequence.

91. A method of developing a biologically active agent that modulates acell signaling event associated with a disease gene, comprising:

a. contacting a test compound with a model cell of any one of paragraphs86-90; and

b. detecting a change in a readout that is indicative of a reduction oran augmentation of a cell signaling event associated with said mutationin said disease gene, thereby developing said biologically active agentthat modulates said cell signaling event associated with said diseasegene.

92. A recombinant polynucleotide comprising a guide sequence upstream ofa traer mate sequence, wherein the guide sequence when expressed directssequence-specific binding of a CRISPR complex to a corresponding targetsequence present in a eukaryotic cell.

93. The recombinant polynucleotide of paragraph 89, wherein the targetsequence is a viral sequence present in a eukaryotic cell.

94. The recombinant polynucleotide of paragraph 89, wherein the targetsequence is a proto-oncogene or an oncogene.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

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What is claimed is:
 1. A CRISPR-Cas complex-mediated method for theproduction of a multicellular genetically modified non-human animal ormulticellular genetically modified plant, the method comprisingintroducing into a cell of the multicellular non-human animal or plantcontaining one or more target sequences, wherein the target sequence isadjacent to a Protospacer Adjacent Motif (PAM), an engineered,non-naturally occurring CRISPR-Cas vector system comprising one or morevectors comprising: a) a first regulatory element operably linked to oneor more nucleotide sequences encoding one or more CRISPR-Cas systemcomplex guide RNAs that hybridize with the target sequence(s), and b) asecond regulatory element operably linked to a nucleotide sequenceencoding a Type II Cas9 protein, wherein components (a) and (b) arelocated on same or different vectors of the system, and wherein theguide RNA comprises a trans-activating cr (tracr) sequence which is 30or more nucleotides in length, whereby when transcribed, the one or moreguide RNAs form the CRISPR-Cas complex with the Cas9 protein, and theone or more guide RNAs direct sequence-specific binding of the CRISPRcomplex to one or more target sequences, and there is PAM recognition,wherein the Cas9 protein and the one or more guide RNAs do not naturallyoccur together, and whereby the multicellular genetically modifiednon-human animal or multicellular genetically modified plant isproduced, and displays a phenotype or carries DNA to display a phenotypeof the genetic modification.
 2. The method according to claim 1, whereinthe Cas9 protein comprises one or more mutations in a catalytic domain,whereby the Cas9 protein is a nickase.
 3. The method according to claim1, wherein the one or more vectors comprise one or more viral vectors.4. The method according to claim 3, wherein the one or more viralvectors comprise one or more retroviral, lentiviral, adenoviral,adeno-associated or herpes simplex viral vectors.
 5. The methodaccording to claim 1 wherein the Cas9 protein comprises one or moremutations in the RuvC I, RuvC II or RuvC III catalytic domains.
 6. Themethod according to claim 1 wherein the Cas9 protein comprises amutation selected from the group consisting of D10A, H840A, N854A andN863A with reference to the position numbering of a S. pyogenes Cas9protein.
 7. The method according to claim 1, wherein the CRISPR-Cascomplex further comprises one or more nuclear localization signal(s)(NLS(s)).
 8. The method according to claim 1, wherein the one or moreguide RNAs comprise a guide sequence fused to the tracr sequence.
 9. Themethod according to claim 1, wherein the one or more guide RNAs comprisea chimeric RNA comprising a guide sequence, the tracr sequence, and atracr mate sequence.
 10. The method according to claim 1, whereinhybridization between the tracr sequence and a tracr mate sequenceproduces a transcript having a secondary structure.
 11. The methodaccording to claim 10, wherein the secondary structure comprises ahairpin.
 12. The method according to claim 1, wherein the cell of themulticellular non-human animal or plant is a mammalian cell.
 13. Themethod according to claim 1, wherein the nucleotide sequence encodingthe Cas9 protein is codon optimized for expression in a eukaryotic cell.14. The method according to claim 1, wherein sequence-specific bindingof the CRISPR complex to one or more target sequences and PAMrecognition obtains cleavage of a polynucleotide loci, and the methodincludes introducing a template into the cleaved polynucleotide loci.15. A CRISPR-Cas complex-mediated method for the production of amulticellular genetically modified non-human animal or multicellulargenetically modified plant, the method comprising delivery to one ormore target sequences in a cell of the multicellular non-human animal orplant of: a Cas9 protein; a guide sequence linked to a tracr matesequence; and a tracr sequence; wherein the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence inthe cell, whereby the multicellular genetically modified non-humananimal or multicellular genetically modified plant is produced, anddisplays a phenotype or carries DNA to display a phenotype of thegenetic modification.
 16. The method according to claim 15, wherein saidguide sequence, tracr mate and tracr sequence are comprised within achimeric RNA.
 17. The method according to claim 15, wherein the Cas9directs cleavage of one or two strands at the location of the targetsequence.
 18. The method according to claim 17 wherein Cas9 is mutatedwith respect to a wild type Cas9 enzyme such that the mutated Cas9 lacksthe ability to cleave one strand of a target polynucleotide containing atarget sequence, or lacks the ability to cleave both strands of a targetpolynucleotide containing a target sequence, wherein the Cas9 includesan effector domain that provides cleavage of the target polynucleotide.19. The method according to claim 18, wherein the Cas9 comprises amutation selected from the group consisting of D10A, H840A, N854A andN863A with reference to the position numbering of a S. pyogenes Cas9protein.
 20. The method according to claim 15, wherein the Cas9 enzymecomprises one or more NLS at the amino-terminus, or one or more NLS atthe carboxy-terminus, or a combination thereof.
 21. The method accordingto claim 15, wherein the Cas9 enzyme is S. thermophilus Cas9.
 22. Themethod according to claim 15, wherein the tracr sequence is 30 or morenucleotides in length.
 23. The method according to claim 15, wherein thetracr sequence is modified.
 24. The method according claim 15, whereinsaid tracr sequence comprises all or a portion of a wild-type tracrsequence.
 25. The method according to claim 15, wherein the Cas9 enzymeincludes one or more heterologous protein domains.
 26. A CRISPR-Cascomplex-mediated method for the production of a multicellulargenetically modified non-human animal or multicellular geneticallymodified plant, the method comprising delivery to a cell of themulticellular non-human animal or plant having one or more targetsequences of a Cas9 protein, or a nucleic acid molecule encoding theCas9 protein; and a guide sequence linked to a tracr mate sequence; anda tracr sequence, or one or more nucleic acid molecules encoding theguide sequence linked to the tracr mate sequence and the tracr sequence,wherein the guide sequence directs sequence-specific binding of a CRISPRcomplex to the target sequence in the cell, whereby the multicellulargenetically modified non-human animal or multicellular geneticallymodified plant is produced, and displays a phenotype or carries DNA todisplay a phenotype of the genetic modification.
 27. The methodaccording to claim 26, wherein said guide sequence, tracr mate and tracrsequence are comprised within a chimeric RNA.
 28. The method accordingto claim 26, wherein the Cas9 directs cleavage of one or two strands atthe location of the target sequence.
 29. The method according to claim28 wherein Cas9 is mutated with respect to a wild type Cas9 enzyme suchthat the mutated Cas9 lacks the ability to cleave one strand of a targetpolynucleotide containing a target sequence or lacks the ability tocleave both strands of a target polynucleotide containing a targetsequence, wherein the Cas9 includes an effector domain that providescleavage of the target polynucleotide.
 30. The method according to claim29, wherein the Cas9 comprises a mutation selected from the groupconsisting of D10A, H840A, N854A and N863A with reference to theposition numbering of a S. pyogenes Cas9 protein.
 31. The methodaccording to claim 26, wherein the Cas9 enzyme comprises one or more NLSat the amino-terminus, or one or more NLS at the carboxy-terminus, or acombination thereof.
 32. The method according to claim 26, wherein theCas9 enzyme is S. thermophilus Cas9.
 33. The method according to claim26, wherein the tracr sequence is 30 or more nucleotides in length. 34.The method according to claim 26, wherein the tracr sequence ismodified.
 35. The method according claim 26, wherein said tracr sequencecomprises all or a portion of a wild-type tracr sequence.
 36. The methodaccording to claim 26, wherein the Cas9 enzyme includes one or moreheterologous protein domains.
 37. The method of claim 26 wherein thenucleic acid molecules are located on the same vector.
 38. The methodaccording to claim 26, wherein the tracr sequence and the tracr matesequence are contained within a single transcript, such thathybridization between the two produces a secondary structure which is ahairpin.
 39. The method according to claim 26, wherein two or more guidesequences or nucleic acid molecule(s) encoding two or more guidesequences are delivered to the cell, each of the two or more guidesequences direct sequence specific binding of a CRISPR complex to adifferent target sequence in the cell.
 40. The method according to claim37, wherein the vector is a plasmid or viral vector.
 41. The methodaccording to claim 40, wherein the viral vector is a retrovirus,lentivirus, adenovirus, adeno-associated virus, or herpes simplex virusvector.